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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

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.......

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 Candid.......

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 .......

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
15 mL conical centrifuge tubesBD Falcon546021
1x PBS-Prepared in labNaCl : 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 tubesBD Falcon546041
96-well microtiter platesNunc442404
IncubatorGeneric
MenadioneSigmaM5625
Microtiter Plate ReaderGeneric
Multichannel pipette and tipsGeneric
Petri dishesTarson460090
Ringers Lactate-Prepared in labsodium 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 MOPSHimediaAT180
Sabouraud dextrose AgarSRL24613
Sabouraud dextrose BrothSRL24835
XTT InvitrogenX6493

References

  1. Bongomin, F., Gago, S., Oladele, R. O., Denning, D. W. Global and multi-national prevalence of fungal diseases-estimate precision. Journal of Fungi. 3 (4), 57 (2017).
  2. Pappas, P., Lionakis, M., Arendrup, M., Ostrosky-Zeichner, L., Kullberg, B. J. Invasive ca....

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BiofilmCandida TropicalisAntibodyAntifungalMetabolic ActivityTetrazolium based Reduction Assay96 well PlateRPMI 1640 MOPS MediumPBS WashSerum DilutionNo fungus Negative Control

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