Antibacterial activity of fungal isolates from the International Collection of Microorganisms from Plants (ICMP) against Mycobacterium abscessus and Mycobacterium marinum.
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.
