Oleic albumin dextrose catalase

mycobacterium avium subsp. paratuberculosis A1-157, an isolate belonging to a dominant clade containing more than 80% of all Canadian MAP isolated (Ahlstrom et al., 2015 (link)) was used as the parental strain for transposon mutant library creation. Bacteria were grown on an orbital shaker at 200 rpm at 37°C in Difco Middlebrook 7H9 media supplemented with 10% OADC (Oleic Albumin Dextrose Catalase, Becton Dickinson and Co., Sparks, MD, USA), 2 g/L mycobactin J (Allied Monitor Inc., Fayette, MO, USA) and 0.4% glycerol. Mycobacterial phage MycomarT7, containing mariner class transposon element and kanamycin resistance cassette was used for transducing the transposon into the MAP A1-157 bacterial cells (Siegrist and Rubin, 2009 ). Mutants were selected on Difco Middlebrook 7H11 plates containing kanamycin (50 μg/ml) and grown for 8 weeks at 37°C in Ziploc bags to prevent them from drying out.
From each library, about 5,000 colonies grew evenly on 20 plates with a diameter of 200 mm. Ahead of plating, potential clumps were broken up by passing the transduced MAP cells through a 30 g needle 10 times. A section per plate was counted after the plates were divided into 8 sections. A total of 100,000 colonies were estimated to be pooled to build one library and three libraries were combined to grow the inoculum.

M. bovis BCG Pasteur strain 1172 P2 (Pasteur Institute, Paris, France) was cultured and grown to the log phase in 7H9 Middlebrook medium. Bacteria were stored at −80 °C in PBS plus 10% glycerol until use. The mycobacterial load was determined by plating serial 10-fold dilutions on agar 7H10 Middlebrook, supplemented with Oleic Albumin Dextrose Catalase (OADC, BD, Franklin Lakes, NJ, USA). BCG colonies were counted after incubation for at least 3 weeks.

M. smegmatis strains were grown in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma) and 10% albumin-dextrose-catalase (BD Diagnostics) at 37°C on a shaking platform or on Middlebrook 7H11 agar supplemented with 10% oleic-albumin-dextrose-catalase (BD Diagnostics) at 37°C for 3 days. For plasmid selection, antibiotics were used at the following concentrations: kanamycin 25 μg/ml (Sigma), hygromycin 50 μg/ml (Roche) and aTc 2-200 ng/ml (Sigma). Bacterial growth was measured by monitoring the OD₆₀₀ over time and viability measurements were made using the Miles and Misra technique; spotting 20 μl of a series of 10-fold dilutions of culture onto agar (Miles et al., 1938 (link)).

Mtb H37Rv was supplied by R.L. Friedmann (University of Arizona, Tucson, AZ). BCG, MDR-Mtb (KMRC-00116-00150), and Mav (ATCC 25291) were acquired from the Korean Mycobacterium Resource Center in the Korean Institute of Tuberculosis (Osong, South Korea). Mycobacteria were cultured in Middlebrook 7H9 (Difco, 271310) medium supplemented with 10% oleic albumin dextrose catalase (OADC; BD Biosciences, San Diego, CA, 212240), 0.5% glycerol, and 0.05% Tween-80 (7H9-OADC) on a rotary shaking incubator (140 rpm) at 37°C to an OD₆₀₀ of 0.4–0.6. Mtb expressing red fluorescent protein (Mtb-ERFP) was cultivated in 7H9-OADC supplemented with 50 µg/ml kanamycin (Sigma-Aldrich, St. Louis, MO, 60615). Bacterial cultures were harvested, and the pellets were washed with phosphate-buffered saline (PBS; LPS solution, Daejeon, Korea, CBP007B) by sequential centrifugation at 2090×g (3000 rpm) for 30 min. To separate bacteria into single cells, pellets resuspended in PBS with 0.1% Tween-80 were subjected to repeated rounds of sonication. The resulting bacterial suspensions were aliquoted and stored at −80°C. Colony-forming units (CFUs) were counted on Middlebrook 7H10 agar (Difco, 262710).

Mabc (ATCC 19977) and Mmass (KMRC-00136-13018) were incubated at 37 °C using an orbital shaker in Middlebrook 7H9 medium containing 10% oleic albumin dextrose catalase (BD, Franklin Lakes, NJ, USA) until the mid-log phase (OD_{600 nm} = 0.4), as described previously^{18 (link)}.
Cells were inoculated for 2 h with either Mabc or Mmass, washed three times with phosphate-buffered saline (PBS) to remove extracellular bacteria, and incubated in fresh media in a 37 °C humidified atmosphere with 5% carbon dioxide. For mouse infection in vivo, mice were administered Mabc or Mmass intranasally (i.n.; 1 × 10⁷ CFU/mouse). For determination of the bacterial burden, mice were euthanized at the indicated times after NTM infection. The lungs were removed and homogenized using a tissue homogenizer (OMNI TH; Omni International, GA, USA) in PBS with 0.1% Tween® 20, as described previously^{18 (link)}.
For analysis of bacterial viability in host cells by CFU assays, infected cells were lysed in distilled water to release intracellular bacteria. Thereafter, the serially diluted homogenates of the infected tissues or cells were inoculated onto 7H10 agar plates, and colonies were counted 3–4 days later.