We screened 36 ICMP fungal isolates for antibacterial
activity against Mycobacterium abscessus and M. marinum. Because of the slow
growth of many mycobacterial species, we routinely use luciferase-tagged
strains for our assays. M. abscessus BSG301 and M. marinum BSG101 (Dalton et al, 2017) are
stable bioluminescent derivatives transformed with the integrating plasmid
pMV306G13ABCDE (Andreu et al, 2010). 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 (Andreu et al, 2012; Dalton et al, 2016) or in vivo
using sensitive imaging equipment (Andreu et al, 2013).
We grew fungal isolates on Potato Dextrose Agar (PDA) prior
to screening for antibacterial activity using a 24 well plate assay. Briefly,
we added 0.5 mL aliquots of agar to triplicate wells of a black 24 well plate and
allowed them to set. In addition to PDA, this comprised: Czapek Solution Agar
(CSA), Czapek Yeast Extract Agar (CYA), Malt Extract Agar (MEA), Malt Yeast
Extract Agar (MYA), Oatmeal Agar (OA), Rice Extract Agar (REA), and Tryptone
Yeast Extract Agar (TYA). With the aid of a sterile scalpel blade, we sectioned
fungal isolates grown on PDA into cubes ≤
5 mm in diameter, and then transferred the cubes to the agar-filled wells of
the 24-well plates ensuring that each cube was placed fungus-side down and
touching the agar. We covered the inoculated 24-well screening plates, sealed
them with parafilm, and incubated them at room temperature.
We monitored fungal growth visually at regular intervals and
recorded the time taken for them to either cover the entire well or to stop
visibly growing. At twice this time, we removed a 6 mm plug of agar from each
well using a biopsy punch. To screen for antibacterial activity, we grew mycobacterial
cultures with shaking (200 rpm) in Middlebrook 7H9 broth supplemented with 10%
Middlebrook ADC enrichment media, 0.4% glycerol and 0.05% tyloxapol. We grew M.
abscessus at 37 °C and M. marinum at 28 °C. We resuspended M. abscessus BSG301
and M. marinum BSG101 in 0.8% Middlebrook 7H9 broth supplemented with 10%
Middlebrook ADC enrichment media to a final concentration of 10^7 colony
forming units (CFU)/mL for M. abscessus and 10^8 CFU/mL for M. marinum. With
the aid of a pipette, we pipetted 50 µL of the bacterial-agar mixture into the
cylindrical holes left after removal of the fungal-agar plugs and allowed the
mixture to set. We measured bacterial luminescence at regular intervals using a
Victor X-3 luminescence plate reader (PerkinElmer) with an integration time of
1 s. Between measurements, plates were covered, placed in a plastic box lined
with damp paper towels, and incubated static at 37 °C for M. abscessus and 28
°C for M. marinum.
We measured antibacterial activity as reductions in light
output of our bioluminescent mycobacterial strains over a 72-hour period. We
calculated activity scores by first converting the luminescence measurement at
each time-point into an area under the curve (AUC) value for each well. We then
divided this number by the median AUC of a sterile control plate inoculated and
incubated at the same time as the fungus-containing plates. The negative log of
this value corresponds to the activity score. We define a fungus-media
combination as active/antibacterial if the median activity score is above 1
which corresponds to a >90% reduction in light compared to the control.
Similarly, an activity score above 2 means corresponds to a >99% reduction.
More detailed protocols are available at protocols.io.
