We thank CAPES-Brazil, CNPq-Brazil, FAP/DF for financial support. We are grateful to Dr. Hugo Costa Paes for revising the manuscript.

1. Solutions and Media
<ol>
	<li>Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate buffered saline (PBS), Sabouraud dextrose broth, and Sabouraud dextrose agar as per Table 1.</li>
</ol>
2. Fungal Inoculum Growth Conditions
<ol>
	<li>Store all fungal strains as frozen stocks in 35% glycerol at -80 &#176;C, until needed.</li>
	<li>Perform the following steps before each experiment.
	<ol>
		<li>For Candida albicans strains:
		<ol>
			<li>Thaw a stock vial and transfer 200 &#181;L to 10 mL of Sabouraud dextrose broth in a sterile 50 mL polypropylene tube with a cap and culture overnight at 30 &#176;C with agitation (200 rpm). Remember to incline the tube and leave the cap slightly open for better aeration of the culture.</li>
		</ol>
		</li>
		<li>For Cryptococcus neoformans strains:
		<ol>
			<li>Scrape the frozen surface of a stock vial. Plate the cells onto Sabouraud dextrose agar plates and incubate for 48 h at 30 &#176;C. After visible growth of isolated colonies, keep the plates in the refrigerator (4 &#176;C) sealed with paraffin film for up to 15 days.</li>
			<li>Collect a medium-sized isolated colony from the plate using a sterile toothpick or sterile inoculating loop and inoculate 10 mL of Sabouraud dextrose broth in a sterile 50 mL conical tube. Incubate for approximately 24 h at 30 &#176;C under agitation (200 rpm).</li>
			<li>Do not exceed the 24 h incubation. Remember to incline the tube and leave the cap slightly open for better aeration. It is always good to standardize the growth stage of the cells before each test, since this is an important factor affecting antimicrobial resistance. 
			NOTE: Both fungi were grown at 30 &#176;C for rapid propagation in our experiments, but the temperature can be changed to reflect the aims of the study, for example clinical treatment (37 &#176;C). Most importantly, this initial incubation time should be established beforehand for each fungal isolate and maintained throughout all the tests to ensure reproducibility.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After fungal growth, collect the cells by centrifuging the conical tubes at 1,200 x g for 5 min at room temperature. Discard the supernatant and add 10 mL of PBS.</li>
	<li>Resuspend the cells and centrifuge again at 1,200 x g for 5 min at room temperature.</li>
	<li>Repeat the PBS wash and centrifugation twice more. After the third wash, resuspend the cells in 5 mL (according to the pellet size) of 2X RPMI-1640 medium.</li>
	<li>Prepare 1 mL of a 1:100 or 1:1,000 dilution in a microcentrifuge tube (depending on the turbidity of the cell suspension).</li>
	<li>Aliquot 10 &#181;L of this dilution, place it in a hemocytometer chamber and count the total number of cells in the four corner quadrants under the microscope. Calculate the concentration by the formula: (total cell number/4) x dilution factor x 104 (chamber dilution constant).
	<ol>
		<li>Consider a 100% viability if viability counts and growth conditions have been systematically correlated for the isolate being studied. 
		NOTE: The standardization and quality control of viability counts can be done by back-plating in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) antifungal Minimum Inhibitory Concentration (MIC) method for yeasts10.</li>
		<li>If the growth/viability correlation has not been established for the isolate under study, measure the fungal viability by counting live cells in a hemocytometer with the aid of dyes that selectively color dead cells, such as phloxine B11, trypan blue12, or Janus Green13. Use the fungal cells for this protocol only if the viability of the population at this point is 90% or above. 
		NOTE: Dead/alive dyes may not work well with the fungus of choice. Please test them before use. For example, the trypan blue dye does not work well with C. neoformans.</li>
	</ol>
	</li>
	<li>After that, prepare the cell suspensions in 2X RPMI-1640 medium (2X adjusted inoculum in RPMI-1640 medium). For 96-well plates consider a volume of 5 mL for each plate.
	<ol>
		<li>For all C. albicans strains, prepare a stock cell suspension of 4 x 103 cells/mL. This concentration is 2X of the final cell concentration in each well (2 x 103 cells/mL).</li>
		<li>For C. neoformans strains, prepare a stock culture of cells 2 x 104 cells/mL. This concentration is twice the final cell concentration in each well (1 x 104 cells/mL).</li>
		<li>For other fungi, test with a known antifungal which concentration will be ideal for the particular study in relation to the incubation time. Take metabolism and duplication time of the fungus into consideration.</li>
	</ol>
	</li>
</ol>
3. Peptides (Unknown Agent)
<ol>
	<li>Store the lyophilized peptides at -20 &#176;C, and dissolve them in deionized water before each experiment. The maximum storage time once dissolved in water will depend on the nature of each peptide.</li>
	<li>Prepare aliquots of twice the highest final concentration tested in the assay (2X). Ideally, prepare a small number of aliquots with enough peptide for one time use to avoid freeze-thaw cycles. 
	NOTE: The choice of concentration should be based on literature and the properties of the peptide. It is recommended to start the serial dilutions with approximately 100 &#181;M of the peptide, and then decrease or increase this concentration range depending on the obtained results.</li>
</ol>
4. Reference Antifungals (Positive Controls)
<ol>
	<li>For those diluted in water: prepare a 2X solution of the highest concentration of the analysis (see Section 5: Antifungal Assay for the dilution steps).
	<ol>
		<li>For C. albicans strains, prepare a solution of 128 &#181;g/mL of fluconazole or 128 &#181;g/mL of caspofungin. The plate concentration for both will be 64 &#181;g/mL.</li>
		<li>For C. neoformans strains, prepare a solution of 32 &#181;g/mL of amphotericin B (water-soluble solution). The plate concentration will be 16 &#181;g/mL. 
		NOTE: Normally amphotericin B is diluted in dimethyl sulfoxide (DMSO), since this anti-fungal is poorly soluble in water. However, there are water-soluble amphotericin B preparations commercially available.</li>
	</ol>
	</li>
	<li>For antifungals diluted in an organic solvent: prepare a 100X stock solution in DMSO, then dilute it to 10X in water for use. 
	NOTE: Thus, in the well, the final concentration of DMSO will not exceed 1%. A well where the fungus will grow in media containing 1% DMSO as control is required given that some fungi do not tolerate well this concentration of the solvent. Remember that DMSO is photosensitive, so cover the plate with foil or place it in a dark chamber for the duration of the incubation period.</li>
</ol>
5. Antifungal Assay
NOTE: In vitro antifungal assays are performed based on the broth microdilution susceptibility test from Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with some modifications.
<ol>
	<li>Prepare a two-fold serial dilution of each peptide and control antifungal in 96-well polystyrene microplates to a final volume of 50 &#181;L.
	<ol>
		<li>Using a pipette add 100 &#181;L of the antifungal/peptide in the 2X concentration of the highest desired final concentration in columns 1-3, in row A.</li>
		<li>Using a multichannel pipette, add 50 &#181;L of sterile water into the other wells, in rows B to H.</li>
		<li>Remove 50 &#181;L of the wells with the highest concentration (row A), transfer to the next well of the next concentration (row B) and homogenize.</li>
		<li>Repeat the above steps until the well with the lowest concentration and discard 50 &#181;L of this well (row H). Leave Columns 11 and 12 (rows A, B, C) with only water for either blank control or negative/growth control.</li>
		<li>Add 50 &#181;L of the 2X adjusted inoculum in RPMI-1640 medium to each well (columns 1-3 plus column 11 (negative/growth control)); the final concentration for C. albicans will be 2 x 103 cells/mL, and the final concentration for C. neoformans strains will be 104 cells/mL.</li>
		<li>Prepare blank controls (50 &#181;L water + 50 &#181;L 2X RPMI-1640 medium without cells) and negative/growth control (50 &#181;L water + 50 &#181;L adjusted inoculum 2X RPMI-1640 medium without antifungal), and load into the well plate as described above.</li>
	</ol>
	</li>
	<li>Repeat the procedure for each compound to be tested and the chosen antifungal control (columns 4-10). 
	NOTE: The serial dilutions can be done vertically as the experiment below, or horizontally in the plate (i.e., if the number of dilutions of the given compound/extract that needs to be tested is eight or more).
	<ol>
		<li>If the compounds, reference drugs, or any of its components are photosensitive, perform the assay in reduced light, cover the plates with foil, or place in a dark chamber during incubations.</li>
	</ol>
	</li>
	<li>Consider the following optional recommendations.
	<ol>
		<li>Seal the plate with a clear cover plate that permits gas exchange, as this helps reduce evaporation.</li>
		<li>Place the plate in a humid chamber; it will also help reduce evaporation.</li>
	</ol>
	</li>
	<li>Incubate the plates at 37 &#176;C for 24 h or 48 h for all C. albicans strains, and for 48 h with 200 rpm shaking for C. neoformans. The lower final volume (100 &#181;L) ensures that no spillover occurs. 
	NOTE: No significant difference was observed between MIC readings at 24 h and 48 h for C. albicans strains. Nonetheless, readings at 48 h are easier to visualize.</li>
	<li>Observe all wells, with the assistance of an inverted optical microscope, before the incubation period and after to verify changes in morphology as well as to check for signs of contamination. 
	NOTE: Readings can be done visually and photographed at the end of the experiment. Before each reading, homogenize slightly the plate. The reads can also be performed by measuring its OD at 600 nm, if cell clumps or filamentation are not observed.</li>
	<li>Perform the experiments at least three times on separate dates.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+drug+resistance:+mechanisms,+epidemiology,+and+consequences+for+treatment.">Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment.</a> Am J Med. 125 (1 Suppl), S3-S13 (2012).</li><li>Hancock, R. E., Diamond, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cationic+antimicrobial+peptides+in+innate+host+defences.">The role of cationic antimicrobial peptides in innate host defences.</a> Trends Microbiol. 8 (9), 402-410 (2000).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snake+cathelicidin+from+Bungarus+fasciatus+is+a+potent+peptide+antibiotics.">Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics.</a> PLoS One. 3 (9), e3217 (2008).</li><li>Du, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AaeAP1+and+AaeAP2:+novel+antimicrobial+peptides+from+the+venom+of+the+scorpion,+Androctonus+aeneas:+structural+characterisation,+molecular+cloning+of+biosynthetic+precursor-encoding+cDNAs+and+engineering+of+analogues+with+enhanced+antimicrobial+and+anticancer+activities.">AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.</a> Toxins (Basel). 7 (2), 219-237 (2015).</li><li>Benincasa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+activity+of+five+cathelicidin+peptides+against+clinically+isolated+yeasts.">Fungicidal activity of five cathelicidin peptides against clinically isolated yeasts.</a> J Antimicrob Chemother. 58 (5), 950-959 (2006).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reference Method for Broth Dilution Antifungal Susceptibiliy Testing of Yeasts; Approved Standard -Third Edition. CLSI document M27-A3. , (2008).</li><li>Guilhelmelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Scorpion+Venom-Derived+Antifungal+Peptides+against+Planktonic+Cells+of+Candida+spp.+and+Cryptococcus+neoformans+and+Candida+albicans+Biofilms.">Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.</a> Front Microbiol. 7, 1844 (2016).</li><li>Roongruangsree, U. T., Kjerulf-Jensen, C., Olson, L. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+as+stain+for+fungi.">Trypan blue as stain for fungi.</a> Stain Technol. 31 (3), 115-116 (1956).</li><li>Goihman-Yahr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71 (2), 73-83 (1980).</li><li>Tati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histatin+5-spermidine+conjugates+have+enhanced+fungicidal+activity+and+efficacy+as+a+topical+therapeutic+for+oral+candidiasis.">Histatin 5-spermidine conjugates have enhanced fungicidal activity and efficacy as a topical therapeutic for oral candidiasis.</a> Antimicrob Agents Chemother. 58 (2), 756-766 (2014).</li><li>Petrou, M. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shaking+on+antifungal+susceptibility+testing+of+Cryptococcus+neoformans:+a+comparison+of+the+NCCLS+standard+M27A+medium,+buffered+yeast+nitrogen+base,+and+RPMI-2%+glucose.">Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose.</a> Antimicrob Agents Chemother. 44 (2), 400-404 (2000).</li><li>Beggs, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase+in+relation+to+ketoconazole+and+miconazole+susceptibilities+of+Candida+albicans.">Growth phase in relation to ketoconazole and miconazole susceptibilities of Candida albicans.</a> Antimicrob Agents Chemother. 25 (3), 316-318 (1984).</li><li>Alcouloumre, M. S., Ghannoum, M. A., Ibrahim, A. S., Selsted, M. E., Edwards, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+properties+of+defensin+NP-1+and+activity+against+Cryptococcus+neoformans+in+vitro.">Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.</a> Antimicrob Agents Chemother. 37 (12), 2628-2632 (1993).</li></ol>

The authors have nothing to disclose.

Microdilution tests can analyze the potential antifungal activity of a target compound using small quantities of the compound, and at the same time test it in a range of concentrations. Accordingly, this protocol is recommended as a first step in screening for potential new antifungal compounds. The protocol presented here is based on the M27-A3 protocol, initially designed to aid in the selection of antifungal therapy in clinics, and can be adapted to a variety of new antifungal compounds. Overall, this protocol can foc...

Fungal infections have become an important medical concern in recent decades, having considerably increased mainly due to a rise in the number of immunocompromised individuals such as those undergoing cancer treatment and those living with HIV/AIDS or transplanted organs1,2. However, a very limited array of available antifungal drugs and the increasing number of reports on fungal resistance to them contribute to the major problems regarding the therapeutics of systemic mycoses3.
A potential source of new antifungal compounds are antimicrobial peptides (AMPs), small cationic peptides produced by many organisms as part of their innate immune response to infection4. Nevertheless, the screening method to test these compounds against fungal pathogens is not standardized. Different procedures have been used to assess the antifungal activity of AMPs, sometimes for the same model microorganism5,6,7. These differences and the lack of detail in some protocols complicate comparisons between compounds and hampers reproducibility.
One way to standardize the testing of new drug candidates is to follow guidelines used to define antifungal susceptibility in clinical settings, such as the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines. However, these antifungal sensitivity tests are too restrictive, and do not take into consideration variation in metabolism across species, as they were only established for a few select agents. For example, they do not take into account the metabolic needs of non-fermenting yeasts.
This protocol allows the assessment of activity of prospective antifungal compounds, and is implemented here for the search for antifungal peptides. It is based on the broth microdilution susceptibility test from the CLSI M27-A3 guidelines with modifications that optimize the screening of new compounds8,9. These changes allow for the use of small amounts of compound, variations in temperature or initial inoculum, and different media for optimal pre-test growth, while standardizing the results with the use of reference antifungals as controls. This method, with the use of multi-well culture plates, makes it possible to quickly and reliably screen a large number of compounds.
Due to its inherent flexibility, this protocol can be used with different chemical classes of compounds and against other microorganisms, with few adaptations.

<table><tbody><tr><td>&lt;strong&gt;Media and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td><td>Thermo Fisher</td><td>31800-022</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propane sulfonic acid&lt;em&gt; (MOPS) (o que a gente usa tem um s&amp;oacute;dio, completa o nome dele please)&lt;/em&gt;</td><td>Sigma-Aldrich</td><td></td><td>Use to buffer 2X RPMI medium</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Din&amp;acirc;mica</td><td>1528-1</td><td>137 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium chloride (KCl)</td><td>J.T.Baker</td><td>3040-01</td><td>2.7 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Sodium phosphate dibasic (Na2HPO4)</td><td>Sigma-Aldrich</td><td>V000129</td><td>10 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium dihydrogen phosphate (KH2PO4)</td><td>Sigma-Aldrich</td><td>60230</td><td>2 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>BD Difco Sabouraud dextrose broth</td><td>BD</td><td>238230</td><td></td></tr><tr><td>BD Difco Sabouraud Dextrose Agar</td><td>BD</td><td>210950</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>V000123</td><td>35% for (solu&amp;ccedil;&amp;atilde;o de estoque? Criopreserva&amp;ccedil;&amp;atilde;o?)</td></tr><tr><td>Sterile water</td><td></td><td></td><td>Para dilui&amp;ccedil;&amp;atilde;o das drogas na dilui&amp;ccedil;&amp;atilde;o seriada</td></tr><tr><td>&lt;strong&gt;Antifungal drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Fluconazole</td><td>Sigma-Aldrich</td><td>F8929</td><td></td></tr><tr><td>Caspofungin</td><td>Sigma-Aldrich</td><td>PHR1160</td><td></td></tr><tr><td>&lt;strong&gt;Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tube</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Dish petri</td><td>J.Prolab</td><td>0304-5</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Sterile Solution Reservoir</td><td>KASVI</td><td>K30-208</td><td>Use to pippet the solutions using the multichannel pippet</td></tr><tr><td>&lt;strong&gt;Equipment and other materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optical microscope</td><td>Nikon</td><td>E200MV</td><td></td></tr><tr><td>Centrifugue</td><td>Thermo Fisher</td><td>MegaFuge 16R</td><td></td></tr><tr><td>Incubator</td><td>Ethik Technology</td><td>403-3D</td><td>Set to 37&amp;deg; C</td></tr><tr><td>Shaker</td><td>New Brunswick Scientific</td><td>Excella E25</td><td>Set to 37&amp;deg; C, 200 RPM</td></tr><tr><td>Cell counting chamber, Neubauer</td><td>BOECO Germany</td><td>BOE 13</td><td></td></tr><tr><td>Multichannel pipette</td><td>HTL</td><td>5123</td><td></td></tr></tbody></table>

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.

The MIC is defined as the lowest antimicrobial compound concentration that completely inhibits visible fungal growth at the end of the incubation period. Since the objective of this protocol is to have a fast method to screen potential antifungals, any well with clear media similar to the blank wells is considered a positive result, whereas any well with turbidity analogous to the negative/growth control wells is considered negative. However, if there is an interest in knowing whether a g...

Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

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.

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 ...

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18.7K Views.  University of Brasília. An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

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

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 ...

Immunology and Infection

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

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Environment

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

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/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 ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

Source: de-Souza-Silva, C. M., et al. <a href="https://app.jove.com/v/57127">Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

1. Solutions and Media

	Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and ...

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

Summary

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Chapters in this video

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

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

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Quantifying the Antifungal Activity of Peptides Against Candida albicans

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

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds