Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).
Mycobacterium tuberculosis (Mtb), the causative agent of TB, was the most significant infectious disease killer globally in 20191. To evade host defenses, Mtb subverts the mycobactericidal activity of innate immune cells such as macrophages and dendritic cells (DCs), allowing it to persist intracellularly and replicate2. The lack of an effective vaccine to prevent adult pulmonary TB and the increasing emergence of drug-resistant strains highlight the urgent need for new therapies.
Adjunctive host-directed therapies (HDT) could shorten treatment time and help overcome resistance3. Preclinical assessment of HDT candidates in vitro to determine mycobactericidal activity within macrophages often relies on the quantification of Mtb growth by colony-forming units (CFU) on solid agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. Commercially available automated, broth-based microbial detection systems are more commonly used in clinical microbiology laboratories for detection and drug susceptibility testing of Mtb and other mycobacterial species in clinical specimens4. These instruments measure growth indirectly based on the bacterial metabolic activity leading to physical changes in the culture media (change in CO2 or O2 levels or pressure) monitored over time5. The readout is time to positivity (TPP), which has previously been shown to correlate with Mtb CFU in sputum specimens of TB patients in response to treatment6,7 and in lysates of infected murine lung and spleen8. In addition, liquid culture detection systems have been used to measure the effect of conventional pathogen-directed therapies on the growth of mycobacteria in axenic culture and cultured macrophages9,10. The instrument has also been used to investigate the innate ability of dendritic cells and of alveolar macrophages to control intracellular growth of Mtb11,12. This experimental protocol demonstrates that a liquid culture diagnostic system can be adapted to perform preclinical screening of HDT for TB in cultured macrophages. Compared to CFU enumeration, the main advantage of this technique is that it considerably reduces the experimental labor and time required to quantify intracellular mycobacterial growth/survival. This technique relies on access to an automated culture instrument that can be used to assess intracellular mycobacterial survival in immune cells treated with a broad range of pharmacological reagents targeting cellular functions to boost host immunity.
The authors have used the liquid culture method described in this protocol to monitor Mtb growth in monocyte-derived macrophages and alveolar macrophages and THP-1 cells differentiated with PMA11,16,17. This technique can also be modified for use with non-adherent cells12. More recently, the instrument was also used in preclinical studies to evaluate inhaled all-trans retinoic acid (AtRA) as an HDT for TB…
The authors have nothing to disclose.
This work was funded by Science Foundation Ireland (SFI 08/RFP/BMT1689), the Health Research Board in Ireland [HRA-POR/2012/4 and HRA-POR-2015-1145] and Royal City of Dublin Hospital Trust.
IX51 Fluorescent Microscope | Olympus, Japan | N/A | AFB detection and imaging |
2 mL microtube, flat bottom, screw cap, sterile | Sarstedt, North Carolina, USA | 72.694.006 | Mtb infection of macropahges |
5 mL syringe, Luer lock | BD Biosciences, San Jose, CA, USA | SZR-150-031K | Mtb infection of macropahges/CFU |
50 mL tube, sterile | Sarstedt, North Carolina, USA | 62.547.254 | Mtb infection of macropahges |
all-trans-Retinoic Acid (ATRA) ≥98% (HPLC) | Sigma Aldrich, Missouri, USA | R2625 | Host directed therapy candidate |
BacT/ALERT 3D Microbial Detection System | Biomerieux ( Hampshire, UK) | 247001 | Broth-based colormetric detection system |
BACT/ALERT MP BACT/ALERT MP Nutrient Supplement | Biomerieux ( Hampshire, UK) | 414997 | Broth-based colormetric detection assay |
BACT/ALERT MP culture bottles | Biomerieux ( Hampshire, UK) | 419744 | Broth-based colormetric detection assay |
BD BBL Middlebrook ADC Enrichment, 20 mL | BD Biosciences, San Jose, CA, USA | M0553 | Mycobacterium liquid culture |
BD BBL Middlebrook OADC Enrichment, 20mL | BD Biosciences, San Jose, CA, USA | M0678 | Colony Forming Units |
Cell scraper, 25 cm | Sarstedt, North Carolina, USA | 83.1830 | Harvest of lmacrophage lysates |
Corning Syringe Filter, 0.2 µm | Corning Incorporated, Germany | 431219 | Sterilization of lysis buffer |
Cover glass (borosilicate), 24 x 50 mm, #1.5 thickness | VWR International Limited | 631 – 0147 | |
Cycloheximide, from microbial | Sigma Aldrich, Missouri, USA | C7698 | Colony Forming Units |
Dako Fluorescent Mounting Medium | Agilent Technologies Ireland Limited | S3023 | Antifade mounting medium |
Dulbecco’s Phosphate Buffered Saline | Sigma Aldrich, Missouri, USA | D8537 | Mtb infection of macropahges |
Fetal Bovine Serum, Gibco | Thermo Fisher, Massachusetts, USA | 10270106 | Macrophage cell culture |
Glycerol, Difco | BD Biosciences, San Jose, CA, USA | 228220 | Colony Forming Units |
Hoescht 33342 (bisBenzimide H 33342 trihydrochloride) | Sigma Aldrich, Missouri, USA | B2261 | Nuclear stain |
IFNγ, recombinant human | R&D Systems Inc, Minnesota, USA | 285-IF | Host directed therapy candidate |
Labtek 2-well chamber slide, sterile, Nunc | Thermo Fisher, Massachusetts, USA | TKT-210-150R | Mtb infection of macropahges |
L-Asparagine, anhydrous | Sigma Aldrich, Missouri, USA | A4159 | Colony Forming Units |
Linezolid | Sigma Aldrich, Missouri, USA | PZ0014 | Antibiotic |
Microlance Hypodermic Needle 25 G | BD Biosciences, San Jose, CA, USA | 300400 | Mtb infection of macropahges/CFU |
Middlebrook 7H10 Agar Base | BD Biosciences, San Jose, CA, USA | M0303 | Colony Forming Units |
Middlebrook 7H9 Broth Base | BD Biosciences, San Jose, CA, USA | M0178 | Mycobacterium liquid culture |
Modified Auramine O Stain and Decolourizer | Scientific Device Laboratory, IL, USA | 345-250 | AFB stain |
Paraformaldehyde | Sigma Aldrich, Missouri, USA | 158127 | Mtb infection of macropahges |
Petri dishes, 92 x 16mm (20/bag) | Sarstedt, North Carolina, USA | 82.1473.001 | Colony Forming Units |
Phorbol 12-myristate 13-acetate (PMA) | Sigma Aldrich, Missouri, USA | P8139 | Macrophage cell culture |
Polysorbate 80, Difco | BD Biosciences, San Jose, CA, USA | 231181 | Mycobacterium liquid culture |
RPMI-1640, Gibco | Thermo Fisher, Massachusetts, USA | 52400025 | Macrophage cell culture |
Sterile Cell Spreader, L-Shaped | Fisherbrand, Thermo Fisher, MA, USA | RB-44103 | Colony Forming Units |
T25 TC flask, angled neck, filter cap, sterile, Nunc | Thermo Fisher, Massachusetts, USA | 156367 | Mycobacterium liquid culture |
THP-1 cell line | ATCC, Virginia, USA | ATCC TIB-202 | Macrophage cell culture |