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 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.
<ol>
	<li>Streak C. tropicalis (strain ATCC 750) onto a Sabouraud dextrose (SAB) agar plate.</li>
	<li>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 &#181;L of the glycerol stock into a sterile 250 mL conical flask containing 50 mL of SAB broth medium.</li>
	<li>Incubate C. tropicalis culture in an orbital shaker at 180 rpm at 30 &#176;C for 24-48 h.</li>
	<li>Centrifuge the fungal culture (cells in logarithmic phase) at 2,150 &#215; g for 15 min at 21 &#176;C.</li>
	<li>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.</li>
	<li>Centrifuge again at 2,150 &#215; g for 15 min at 21 &#176;C. Discard the supernatant and resuspend the fungal pellet in 10 mL of sterile 1x PBS.</li>
	<li>Calculate the concentration of cells by counting with a haemocytometer.</li>
	<li>Prepare fungal stocks at a final density of 1.0 &#215; 106 cells/mL in RPMI 1640 morpholinepropanesulfonic acid (MOPS) medium. Use the cell suspension from step 1.6 immediately. 
	&#8203;NOTE: To set up one 96-well plate, the total fungal stock volume needed is 10 mL (100 &#181;L/well). Scale as needed.</li>
</ol>
2. C. tropicalis biofilm formation
<ol>
	<li>Prepare Candida biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1)28,29.</li>
	<li>Add 100 &#181;L 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 &#39;no fungus plus serum&#39; and &#39;no fungus and no serum&#39; negative controls by not adding fungal cells. Fill columns 11 and 12 with 100 &#181;L of RPMI 1640 MOPS medium alone.</li>
	<li>Cover the microtiter plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C under stationary conditions.</li>
	<li>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.</li>
	<li>Wash the plate with 200 &#181;L 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).</li>
	<li>To remove excess PBS, air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet.</li>
</ol>
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.
<ol>
	<li>Perform heat-inactivation of the serum (source of polyclonal antibodies) at 56 &#176;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.</li>
	<li>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.</li>
	<li>Add 100 &#181;L 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).
	<ol>
		<li>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.</li>
		<li>In column 11, add a 1:50 dilution of serum to all wells to serve as the no fungus plus serum negative control.</li>
		<li>In column 12, do not add serum dilution to any well; keep this as the no fungus no serum negative control.</li>
	</ol>
	</li>
	<li>Cover the plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C.</li>
</ol>
4. Biofilm metabolic activity estimation
<ol>
	<li>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.</li>
	<li>Wash the plate with 200 &#181;L 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.</li>
	<li>Preparation of XTT/menadione:
	<ol>
		<li>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 &#176;C.</li>
		<li>Prepare menadione as a 10 mM stock. Dissolve 8.6 mg of menadione in 5 mL of acetone and distribute 50 &#181;L in 100 separate microtubes. Store the aliquots at -80 &#176;C.</li>
		<li>Prepare XTT/menadione solution just before use by taking 10 mL of XTT and adding 1 &#181;L of menadione to obtain a 1 &#181;M working solution.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L 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 &#176;C in the dark.</li>
	<li>Transfer 80 &#181;L of the colored supernatant from each well into a fresh 96-well plate. Read the plate at 490 nm.</li>
	<li>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 - <img alt="Equation 1" src="/files/ftp_upload/64425/64425eq01.jpg" style="margin: auto;" /> &#215; 100&#160; &#160;&#160;(1)</li>
</ol>

The authors declare no conflicts of interest.

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 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 cells33,34. 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 well36,37. 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 studies7,8. 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&#39;s sensitivity, one could decrease the time needed for biofilm to form, increase the antifungal agent&#39;s concentration, or shorten the assay&#39;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,&#160;which can merit the extension of this protocol to the genus Candida16,41,44,45,46,47.
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.

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><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&lt;br/&gt; KCl : 0.1 g&lt;br/&gt; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;:&amp;nbsp; 0.72 g&lt;br/&gt; KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; : 0.12 g&lt;br/&gt; 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&amp;nbsp;</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&amp;nbsp;</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 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 &#8722; 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&#39;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 &#60;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%).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64425/64425fig01.jpg" /> 
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 &#181;m (A,B), 10 &#181;m (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64425/64425fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64425/64425fig02.jpg" /> 
Figure 2: Layout of the 96-well plate format.&#160;Addition of&#160;(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). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64425/64425fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64425/64425fig03.jpg" /> 
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 &#177; SEM (n = 3). Ordinary one-way ANOVA followed by Dunnett&#39;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 &#60; 0.0001. Abbreviations: PBS = phosphate-buffered saline; Sap2 = secreted aspartyl proteinase 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64425/64425fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="min-width: 75px;">&#60;&#62;</td>
			<td style="min-width: 75px;">1</td>
			<td style="min-width: 75px;">2</td>
			<td style="min-width: 75px;">3</td>
			<td style="min-width: 75px;">4</td>
			<td style="min-width: 75px;">5</td>
			<td style="min-width: 75px;">6</td>
			<td style="min-width: 75px;">7</td>
			<td style="min-width: 75px;">8</td>
			<td style="min-width: 75px;">9</td>
			<td style="min-width: 75px;">10</td>
			<td style="min-width: 75px;">11</td>
			<td style="min-width: 75px;">12</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">A</td>
			<td style="min-width: 75px;">0.475</td>
			<td style="min-width: 75px;">0.4141</td>
			<td style="min-width: 75px;">0.0441</td>
			<td style="min-width: 75px;">0.4637</td>
			<td style="min-width: 75px;">0.485</td>
			<td style="min-width: 75px;">0.0484</td>
			<td style="min-width: 75px;">0.5179</td>
			<td style="min-width: 75px;">0.4068</td>
			<td style="min-width: 75px;">0.0442</td>
			<td style="min-width: 75px;">0.8826</td>
			<td style="min-width: 75px;">0.0452</td>
			<td style="min-width: 75px;">0.0404</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">B</td>
			<td style="min-width: 75px;">0.3848</td>
			<td style="min-width: 75px;">0.3773</td>
			<td style="min-width: 75px;">0.0428</td>
			<td style="min-width: 75px;">0.369</td>
			<td style="min-width: 75px;">0.3781</td>
			<td style="min-width: 75px;">0.0465</td>
			<td style="min-width: 75px;">0.4105</td>
			<td style="min-width: 75px;">0.3773</td>
			<td style="min-width: 75px;">0.0447</td>
			<td style="min-width: 75px;">0.8428</td>
			<td style="min-width: 75px;">0.0426</td>
			<td style="min-width: 75px;">0.041</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">C</td>
			<td style="min-width: 75px;">0.2754</td>
			<td style="min-width: 75px;">0.2728</td>
			<td style="min-width: 75px;">0.0427</td>
			<td style="min-width: 75px;">0.305</td>
			<td style="min-width: 75px;">0.3083</td>
			<td style="min-width: 75px;">0.0415</td>
			<td style="min-width: 75px;">0.3531</td>
			<td style="min-width: 75px;">0.3213</td>
			<td style="min-width: 75px;">0.0432</td>
			<td style="min-width: 75px;">0.9661</td>
			<td style="min-width: 75px;">0.0444</td>
			<td style="min-width: 75px;">0.0453</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">D</td>
			<td style="min-width: 75px;">0.6965</td>
			<td style="min-width: 75px;">0.7185</td>
			<td style="min-width: 75px;">0.043</td>
			<td style="min-width: 75px;">0.6681</td>
			<td style="min-width: 75px;">0.6711</td>
			<td style="min-width: 75px;">0.0403</td>
			<td style="min-width: 75px;">0.6346</td>
			<td style="min-width: 75px;">0.728</td>
			<td style="min-width: 75px;">0.0433</td>
			<td style="min-width: 75px;">0.8643</td>
			<td style="min-width: 75px;">0.0384</td>
			<td style="min-width: 75px;">0.0358</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">E</td>
			<td style="min-width: 75px;">0.7031</td>
			<td style="min-width: 75px;">0.7117</td>
			<td style="min-width: 75px;">0.0481</td>
			<td style="min-width: 75px;">0.7806</td>
			<td style="min-width: 75px;">0.688</td>
			<td style="min-width: 75px;">0.0521</td>
			<td style="min-width: 75px;">0.7095</td>
			<td style="min-width: 75px;">0.6998</td>
			<td style="min-width: 75px;">0.0525</td>
			<td style="min-width: 75px;">0.7883</td>
			<td style="min-width: 75px;">0.0491</td>
			<td style="min-width: 75px;">0.0416</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">F</td>
			<td style="min-width: 75px;">0.7588</td>
			<td style="min-width: 75px;">0.7441</td>
			<td style="min-width: 75px;">0.0456</td>
			<td style="min-width: 75px;">0.7173</td>
			<td style="min-width: 75px;">0.7233</td>
			<td style="min-width: 75px;">0.0437</td>
			<td style="min-width: 75px;">0.7733</td>
			<td style="min-width: 75px;">0.6808</td>
			<td style="min-width: 75px;">0.0462</td>
			<td style="min-width: 75px;">0.7256</td>
			<td style="min-width: 75px;">0.0468</td>
			<td style="min-width: 75px;">0.0354</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">G</td>
			<td style="min-width: 75px;">0.7681</td>
			<td style="min-width: 75px;">0.7708</td>
			<td style="min-width: 75px;">0.0435</td>
			<td style="min-width: 75px;">0.7949</td>
			<td style="min-width: 75px;">0.7496</td>
			<td style="min-width: 75px;">0.0445</td>
			<td style="min-width: 75px;">0.7695</td>
			<td style="min-width: 75px;">0.7898</td>
			<td style="min-width: 75px;">0.0451</td>
			<td style="min-width: 75px;">0.7335</td>
			<td style="min-width: 75px;">0.0429</td>
			<td style="min-width: 75px;">0.0371</td>
		</tr>
		<tr>
			<td style="min-width: 75px;">H</td>
			<td style="min-width: 75px;">0.7652</td>
			<td style="min-width: 75px;">0.7855</td>
			<td style="min-width: 75px;">0.0469</td>
			<td style="min-width: 75px;">0.7818</td>
			<td style="min-width: 75px;">0.7766</td>
			<td style="min-width: 75px;">0.0428</td>
			<td style="min-width: 75px;">0.7592</td>
			<td style="min-width: 75px;">0.7765</td>
			<td style="min-width: 75px;">0.0445</td>
			<td style="min-width: 75px;">0.7295</td>
			<td style="min-width: 75px;">0.044</td>
			<td style="min-width: 75px;">0.0341</td>
		</tr>
	</tbody>
</table>
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.

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 ...

a-soluble-tetrazolium-based-reduction-assay-to-evaluate-effect

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.0K Views.  Indian Institute of Technology Roorkee. 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.

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Medicine

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.
Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing.&#160;Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.
In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Biology

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Source: Chandley, P., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

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

Summary

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Microtiter Dish Biofilm Formation Assay

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm