TITLE: Bioluminescence-based 24 well plate assay for screening fungi for activity against Mycobacterium abscessus
AUTHORS: Siouxsie Wiles, Alex Grey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>There is a real and urgent need for new antibiotics which are able to kill Mycobacteria. This group of bacteria includes</span><span style = "font-style:italic;">Mycobacterium tuberculosis</span><span>, a deadly human pathogen that latently infects a third of the world’s population. Multi-drug resistant (MDR) and totally-drug resistant (TDR) isolates have also now evolved. Of increasing importance in many countries are the Non-Tuberculous Mycobacteria (NTM), mycobacterial species which are common soil and water contaminants. Many NTM are able to cause chronic lung and skin and soft tissue infections, especially in patients with suppressed immune systems. One example are members of the </span><span style = "font-style:italic;">Mycobacterium abscessus</span><span> complex. </span><span style = "font-style:italic;">M. abscessus</span><span> infections are difficult to treat due to antibiotic resistance and new compounds that target this group are much needed.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains</span><span style = "vertical-align:super;">1</span><span> for our assays. As bacteria only produce light when alive, bioluminescence is an excellent non-destructive real-time reporter to assay for anti-mycobacterial activity in microtitre plate formats using a luminometer</span><span style = "vertical-align:super;">2-4</span><span>or </span><span style = "font-style:italic;">in vivo</span><span> using sensitive imaging equipment.</span><span style = "vertical-align:super;">5</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>In this protocol, we describe a luminescence-based 24-well plate assay for medium-throughput screening of fungal isolates grown on a variety of media for anti-mycobacterial activity against </span><span style = "font-style:italic;">M. abscessus</span><span>. </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">References:</div></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible UE, Robertson BD, Wiles S (2010). Optimisation of bioluminescent reporters for use with Mycobacteria. PLOS One. 5(5):e10777 (doi:10.1371/journal.pone.0010777).</li><li style = "counter-reset:ol0;">Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD (2012). Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. Journal of Antimicrobial Chemotherapy. 67(2): 404-14 (doi: 10.1093/jac/dkr472). </li><li style = "counter-reset:ol0;"><span>Dalton JP, Uy B, Phummarin N, Copp BR, Denny WA, Crosier PS, Swift S, Wiles S (2016). Effect of common and experimental anti-tuberculosis treatments on </span><span style = "font-style:italic;">Mycobacterium tuberculosis</span><span> growing as biofilms. PeerJ. 4:e2717 (doi: 10.7717/peerj.2717).</span></li><li style = "counter-reset:ol0;">Dalton JP, Uy B, Okuda K, Hall CJ, Denny WA, Crosier PS, Swift S, Wiles S (2017).Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model. Journal of Antimicrobial Chemotherapy 72(2):421-427 (doi: 10.1093/jac/dkw421).</li><li style = "counter-reset:ol0;"><span>Andreu N, Zelmer A, Sampson SL, Ikeh M, Bancroft GJ, Schaible UE, Wiles S, Robertson BD (2013). Rapid in vivo assessment of drug efficacy against </span><span style = "font-style:italic;">Mycobacterium tuberculosis</span><span> using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy. 68(9):2118-27 (doi: 10.1093/jac/dkt155).</span></li></ol></div></div>

[STEPS]
?. [Initial culturing of fungi]
Sub-culture your fungus of choice onto a Potato Dextrose Agar (PDA) plate from either an existing agar culture or from frozen stocks.
?. [Initial culturing of fungi]
Seal the plate with parafilm and store it in a plastic box at room temperature. Allow the culture to reach at least 50% growth for sub-culturing on to well plates for screening. Fungi can take days to months to reach maximal growth.
?. [Preparation of 24 well plates]
Sterilise your 24-well plates and lids using UV irradiation in a biological safety cabinet for at least an hour.
?. [Preparation of 24 well plates]
Dispense 0.5 mL of each agar type into the three wells each of a 24-well plate using a serological pipette. Our plate layout is shown in Figure 1.
?. [Preparation of 24 well plates]
Allow the agar to set.
?. [Preparation of 24 well plates]
Store the plates at 4 °C.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Set the Germinator to 250 °C or higher. Leave the germinator on for at least 30 minutes to ensure that it reaches the appropriate temperature.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Attach the scalpel blade to the handle.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Sterilise the blade using the Germinator.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Using the sterile scalpel blade, section the agar culture using the scalpel blade into cubes ≤ 5 mm in diameter.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Use the scalpel blade to transfer the cubes of fungus into each agar-filled well of a 24-well plate. Ensure that each cube is touching the agar in the well and is inoculated fungus-side down.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Replace the lid and parafilm the edge of the plate.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Repeat for each fungal isolate. When inoculating more than one isolate, work with one isolate at a time, and sterilise the hood with 10% Trigene when switching between each to prevent cross-contamination.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Attach masking tape to the lid of each sterile 24-well plate and label it with your name, the date, and the fungus you inoculated the well plate with.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Store plates in a labelled plastic box. Store them agar-side down initially to ensure that the cubes don’t fall off the agar but flip them over once the fungi have shown visible growth to reduce the chances of contamination.
?. [Culturing fungi - carry this out in a biological safety cabinet if possible to prevent contamination.]
Check your cultures at least twice a week for growth and contamination. If the growth rate differs between wells with different culture media, use the growth in PDA as a reference. We aim to screen each fungus when it reaches 200% growth. Record the age at which it either covers the entire well or stops visibly growing and screen it at double that age. If a well is contaminated, you don’t need to throw the whole plate away. Instead, use a sterile spatula, pipette tip, or similar instrument to scoop out the agar containing the contaminated culture. Discard the contaminated agar and dispose of using the appropriate procedure. Make sure you record which well(s) you have removed.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Record the date, the fungal isolates you are screening, and the age of these cultures. If any wells were removed, make sure you record the location of these wells.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Melt 0.8% 7H9 agar in the microwave and cool it back down to 50 °C in a water bath.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Transfer 2.7 mL aliquots of 0.8% 7H9 agar into your 5 mL tubes. You will later supplement these with 300 μL OADC to make up a final volume of 3 mL. This is enough to comfortably fill all two 24 well plates.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
In a biological safety cabinet, use a biopsy punch to remove the centre of each well culture. Repeat for each fungus you want to test, as well as an extra uninoculated well plate, which will be used as a control. Discard these agar plugs in an empty Petri dish.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Measure the optical density of the bacterial culture at 600nm (OD600). To do this we generally dilute the bacterial culture 1:10 in a 1.5 mL cuvette with MHB (720 µL broth + 80 µL bacteria).
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Dilute the bacterial culture with supplemented 7H9 broth to give a final OD600 of 1.0 which is the equivalent of ~109bacteria per mL.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Dilute your readjusted culture of M. abscessus 1:100 into the 7H9 agar. We usually add 30 μL of bacteria into 3 mL of agar per 5 mL tube. Vortex the mixture for 10 seconds. Use a pipette to dispense 50 µL of the agar mixture into the cylindrical holes left after the fungal plugs were removed.
?. [Inoculation of 24 well screening plates with Mycobacterium abscessus]
Repeat for all well plate cultures and allow the pipetted agar to set.
?. [Checking the bacterial inoculum]
Add 90 µL of MHB or Phosphate Buffer Solution (PBS) to each of the wells in a single column of a 96 well plate. Mix in 10 µL of bacteria inoculum to the top well, mix and discard the tip. Using a clean tip, remove 10 µL, add it to the next well in the column and mix. Repeat to perform a 10-fold serial dilution down the column, using a clean tip each time.
?. [Checking the bacterial inoculum]
Plate three technical replicates of each dilution onto a Middlebrook 7H11 agar plate. Incubate agar plates upside down at 37 °C until visible colonies appear. For M. abscessus this usually takes approximately 3 – 5 days.
?. [Checking the bacterial inoculum]
Count visible colonies to ensure inoculum was correct at approximately 107 CFU/mL.
?. [Measuring bacterial bioluminescence]
We use a Perkin Elmer Victor X plate luminometer set to read 24 well plates with an integration time of 1 second per well. Measure all 24 wells of each plate, even if some have been removed and aren’t inoculated with bacteria.
?. [Measuring bacterial bioluminescence]
We take measurements immediately after setting up the plate (T0) and then at 24 (T24), 48 (T48), and 72 (T72) hours.
?. [Measuring bacterial bioluminescence]
Between measurements, place lids on the plates, put in a plastic box lined with damp paper towels, and incubate at 37 °C without shaking.