TITLE: Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of fungal extracts against Mycobacterium marinum
AUTHORS: Siouxsie Wiles, Alex Grey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><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. Due to the difficulties and dangers involved in culturing </span><span style = "font-style:italic;">M. tuberculosis</span><span>, an airborne Biosafety Level 3 pathogen, faster-growing, and less pathogenic mycobacterial species are routinely exploited for TB research and anti-mycobacterial drug discovery. </span><span style = "font-style:italic;">M. marinum </span><span>shares conserved virulence determinants with </span><span style = "font-style:italic;">M. tuberculosis</span><span> and is a pathogen of ectotherms (fish, amphibians, and reptiles), as well as causing granulomatous skin infections in humans.</span></div><div class = "text-block">Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains (1) 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 (2-4) or in vivo using sensitive imaging equipment (5). This protocol is modified from the method described by Dalton et al (4).</div><div class = "text-block">References:</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]
?. [Preparing 96 well plates]
We test doubling dilutions of each extract fraction in duplicate with a maximum concentration of 1000 µg/mL (Fig. 1A). Each round of screening also requires control wells containing a series of dilutions of the solvent used (we use DMSO), an antibiotic(we use rifampicin at a maximum concentration of 10 µg/mL when testing against M. marinum), and a broth control (Fig. 1B). Using the plate layout described in Figure 1, each 96 well plate can contain either 1 complete set of a crude extract and its 5 fractions or the appropriate controls for a testing round on a separate plate. Record the code of each extract that you are testing in your lab book.
?. [Preparing 96 well plates]
Set up your plate according to Figure 1. Use masking tape to make a front label on the lid including your name or initials, the date, the details of the bacteria being tested, and the extract being tested.
?. [Preparing 96 well plates]
The final volume you will be using in each well is 100 µL. Add 50 µL of supplemented Mueller Hinton Broth (MHB) to all the wells on the first plate except the top row (A).
?. [Preparing 96 well plates]
Add all extracts at double their required concentration to wells in the top row of the plate. If the extracts were dissolved at 50 mg/mL, add 96 µL MHB to wells in the top row and 4 µL of each extract fraction. Mix well by gently aspirating up and down using a pipette and make sure to change pipette tips between extract fractions. For the controls, add 96 µL supplemented MHB to wells in the top row and 4 µL of DMSO to the “DMSO” column, and 2 µL of rifampicin to the “Antibiotic” column. Add 100 µl supplemented MHB to the “Broth” column.
?. [Preparing 96 well plates]
Using a multichannel pipette, gently aspirate repeatedly to homogenise the wells of each row. Then transfer 50 µL from the first row to the second row and aspirate to mix. Discard tips and repeat the doubling dilution down the plate, changing tips between rows, until you reach row G. Do not continue the dilution into row H.
?. [Preparing 96 well plates]
Aspirate 50 µL from the wells in row G and discard the solution. This will leave row H as a growth control containing no extract/DMSO/antibiotic.
?. [Adding the bacteria]
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).
?. [Adding the bacteria]
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). Dilute the bacterial culture with supplemented MHB to give a final OD600of 0.001 which is the equivalent of ~106 bacteria per mL. We do this by diluting the bacterial culture to an OD600 of 0.01 and then diluting that 1 in 10 to give 0.001.
?. [Adding the bacteria]
Calculate the volume of bacteria you will need for all the plates you have set up. You will need 50 µL in all wells to get to 100 µL total volume. Calculate the number of total wells and multiply by 50 µL, for example, 128 wells x 50 µL = 6400 µL or 6.4 mL (V2). Add some extra to account for any pipetting errors and to check the actual inoculum by plating out for colony forming units (CFU).
?. [Adding the bacteria]
We usually make up our bacterial inoculum in a 50 mL Falcon tube using a serological pipette to add the appropriate volume of supplemented MHB.
?. [Adding the bacteria]
Tip the bacterial inoculum into a pipetting reservoir and use a multichannel pipette to add 50 µL to all the wells excluding the MHB sterile control column.
?. [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 bacterial 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]
Count visible colonies to ensure inoculum was correct at approximately 106CFU/mL.
?. [Checking the bacterial inoculum]
Plate three technical replicates of each dilution onto a Middlebrook 7H11 agar plate supplemented with 0.4% glycerol and 10% Middlebrook OADC growth supplement. Incubate agar plates upside down at 28 °C until visible colonies appear. For M. marinum this usually takes approximately 7 – 10 days.
?. [Measuring bacterial bioluminescence]
We use a Perkin Elmer Victor X plate luminometer set to read 96 well plates with an integration time of 1 second per well. If we haven’t filled the entire plate, we change the settings so that the machine doesn’t measure the empty wells.
?. [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 28 °C with shaking at 100 RPM.
?. [Measuring bacterial bioluminescence]
After the final time-point, if the light has reduced to background levels in any wells (for our machine this is