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 experiments outlined in this protocol were carried out using the attenuated H37Ra strain of Mtb, which can be handled in a Containment Level 2 laboratory. All manipulations of live mycobacteria were carried out in Class II biological safety cabinet (BSC). Experimental procedures were designed to minimize the generation of aerosols. Eukaryotic cell culture (THP-1 cells) was also carried out in a Class II BSC. The laboratory carried out a risk assessment and ensured that all procedures were carried out in line with institutional and national biological safety regulations. The human monocytic THP-1 cell line was used to perform the method as described (step 1). Cells are differentiated into macrophages following stimulation with phorbol 12-myristate 13-acetate (PMA) before infection with mycobacteria.
1. Cell culture
2. Quantification of Mtb uptake
3. Harvesting samples for the liquid culture detection system
NOTE: On the day of infection, extracellular mycobacteria are removed by washing, and intracellular mycobacteria are harvested by lysis of one well of macrophages (3 h sample) to determine the initial amount phagocytosed as a baseline control for infection. At subsequent times both the medium, cell lysate, and washes are combined to measure total mycobacterial growth. Extracellular and intracellular growth can also be assessed separately if desired.
The automated liquid culture instrument used in this study monitors CO2 levels every 10 min. A color change in the sensor at the bottom of the instrument bottle is measured colorimetrically and expressed as reflectance units. The instrument software then applies detection algorithms to calculate time to positivity (TTP), i.e., the number of days from inoculation until cultures are flagged as positive (Figure 1A). An inverse relationship between TTP and log10CFU (determined by the agar plate method)12 in the initial inoculum is illustrated in Figure 1B. When Mtb growth within macrophages-infected at MOI ranging from 1-2 to 5-10 in the presence or absence of pharmacological inhibitors (Figure 1C) was compared by the automated culture method described or by enumeration of colonies on solid agar, there was a significant correlation between results obtained by both methods (Figure 1D). To display Mtb growth graphically, the percentage change in TTP was calculated according to the equation above by comparing the TTP values for up to 8 days after infection of macrophages to the initial TTP value12. Results demonstrated a similar trend between liquid culture and CFU, showing significant inhibition of Mtb growth in macrophages in the presence of AtRA in solution or the equivalent dose of AtRA encapsulated in PLGA microparticles (MP) (Figure 2A,B).
The efficacy of another HDT, IFNγ, which has been used to treat multi-drug resistant TB14, in combination with the second-line antibiotic linezolid, was also investigated. A dose-response experiment was first carried out in infected THP-1 cells to determine the efficacy of linezolid alone at concentrations ranging from 0.6-5.0 µg/mL (Figure 2C) before the combination of linezolid at a suboptimal dose (1.25 µg/mL) and IFNγ was tested: there was no significant interaction between the drugs (Figure 2D).
Figure 1: Quantification of Mtb growth by automated liquid culture and on solid medium. Mtb H37Ra was diluted in Middlebrook broth over a range of dilutions (1:2 to 1:100,000). A 300 µL aliquot of each dilution was injected into duplicate instrument culture bottles, incubated on the liquid culture instrument, and their growth was monitored. Simultaneously, an aliquot (10 µL) was spread on MB agar plates containing 0.5% glycerol and 10% OADC (Oleic acid, albumin, dextrose, catalase supplement) in triplicate, for CFU enumeration as previously published12. (A) Data points from the liquid culture system of each dilution are plotted as reflectance units versus time15. To allow for comparisons between samples, the background was normalized to the 9 h reflectance reading for each sample. Growth was monitored for 42 days in all (the first 21 days is shown on graph), time to positivity (TTP) ranged from 3.83 to 11.1 days. (B) TTP from diluted samples was plotted against log10 CFU estimation; each data point represents the values for two replicates from one experiment and describes two separate experiments. The line of best fit and regression coefficient (r2) are shown. (C) Example of AFB staining of intracellular Mtb H37Ra in THP-1 macrophages with auramine (green) which is used to calculate multiplicity of infection, nuclei are counterstained with Hoechst 333258 (blue). Images were generated using an Epifluorescent Microscope with a 100x (numerical aperture [NA], 1.3) oil objective. Scale bar represents 2 µm. (D) Correlation analysis (Pearson) of paired TTP (liquid culture) and CFU estimation of Mtb H37Ra growth in THP-1 macrophage lysates from multiple experiments (n = 23), treated with/without pharmacological reagents and infected at MOI ranging from 1-2 to 5-10 bacilli/infected macrophage. Please click here to view a larger version of this figure.
Figure 2: Evaluation of host-directed therapies for TB using an automated liquid culture system. THP-1 macrophages were infected with Mtb H37Ra for 3 h, extracellular bacteria were removed, and cells were treated with candidate host-directed therapies for up to 192 h. (A) CFU estimation of Mtb growth in macrophages treated with AtRA solution (Sol) or 2 µM AtRA microparticles (MP) (10-20 µg/mL). (B) % change in (time to positivity TTP) in macrophages treated with AtRA solution or AtRA PLGA MP. Infected THP1 cells cultured in 0.1% DMSO in cRPMI were designated as untreated controls. (C) Macrophages were treated with increasing concentrations of linezolid solution (0.6 µg/mL to 5 µg/mL), % change in TTP was determined 24 and 72 h post-infection using an automated liquid culture system. (D) Macrophages infected with Mtb H37Ra were treated with linezolid alone (1.25 µg/mL) or linezolid + IFNγ (5 ng/mL) up to 72 h, % change in TTP was calculated. Untreated controls consisted of infected THP1 cells cultured in cRPMI alone for the indicated times. Please click here to view a larger version of this figure.
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 TB17. Critical steps in the protocol include (1) controlling for uptake of Mtb by AFB staining to mitigate factors such as differential phagocytosis due to drug pre-treatment and minimize cell death which is less likely to occur at lower MOI, (2) depending on the MOI, cell lysates may need to be diluted to maximize the dynamic range of the assay. Aiming for a TTP of approximately 5-12 days has yielded reproducible results.
In this protocol, attenuated Mtb H37Ra has been used as a surrogate for virulent Mtb, but the method can also be applied to study drug efficacy against virulent laboratory and clinical strains in a Containment Level 3 facility with appropriate modification. Confirmation of these results in a mouse model of virulent TB whereby inhaled AtRA, both in solution and encapsulated in poly(lactic-co-glycolic acid (PLGA) microparticles (MP), significantly improved clearance of Mtb-H37Rv from the lung while reducing pathology, indicates that H37Ra is a helpful surrogate for virulent Mtb in these studies17.
As HDTs are designed to be used as adjuncts to conventional therapeutics, it is essential to evaluate their efficacy in vitro in combination with anti-tubercular antibiotics to analyze potential drug interactions18. To obtain a measurable TTP, antibiotics-particularly those with high intracellular efficacy-must first be titrated to suboptimal concentrations (below the minimum inhibitory concentration) before evaluating HDT/antibiotic combinations in macrophages. In the example shown in this protocol (Figure 2C,D), linezolid alone was first titrated before being evaluated in combination with IFN-γ. In another example, lysates of H37Ra-infected THP-1 macrophages treated with rifampicin at or above 0.6 µg/mL do not reliably turn positive in the liquid culture system17. Therefore, a lower concentration of rifampicin is needed to evaluate any HDT combined with this antibiotic in macrophages.
Due to the tendency of Mtb to form aggregates, it is necessary to break up clumps to provide a single cell suspension to achieve uniform aliquoting of mycobacteria into multiple wells of cultured macrophages in each experiment. Similarly, this disaggregation step is repeated after macrophages have been lysed to provide an accurate estimation of growth. In the present protocol, the mycobacteria are passed through a 25 G needle using a syringe to generate a single-cell suspension. Other dispersal methods include sonication of the mycobacterial suspension in a sonicating water bath or vortexing with glass beads19. However, both of these techniques can alter the composition of the outer capsule of Mtb20,21 and may influence binding to and phagocytosis by macrophages21.
Pharmacological compounds can influence the uptake of mycobacteria if they are incubated with macrophages before or during infection. In addition, when performing experiments with primary human macrophages, there may be variation in phagocytosis of mycobacteria between donors. In these circumstances, using a fixed ratio of mycobacteria: macrophages would lead to differences in uptake during the 3 h infection period. This may lead to erroneous assumptions about the efficacy of compounds. This can be avoided by performing staining for acid fast bacilli (AFB) using the auramine method described here (or another AFB stain) in the presence of each HDT and adjusting the initial inoculum accordingly22. While prone to a certain amount of error due to difficulty distinguishing extracellular from intracellular mycobacteria19 and the likelihood that small clumps are still present, the AFB staining procedure helps to equalize the initial level of intracellular infection for each treatment.
In this protocol, for samples collected at times other than the initial 3 h infection sample, the lysate and supernatant are combined to collect intracellular mycobacteria and those that have been released extracellularly. The reason for this is that, since the macrophages have been washed after the initial 3 h incubation with Mtb, extracellular mycobacteria present are likely to have been released by necrotic macrophages. On the other hand, if investigators prefer to harvest the remaining intracellular mycobacteria and exclude extracellular mycobacteria, this method is easily adapted to do so.
Advantages of automated liquid culture-based instruments compared to the solid medium are simplicity of set up, a more efficient experimental workflow as analysis of serial dilutions is unnecessary, an accurate readout is obtained compared to the more subjective manual counting of colonies, faster time to results (approximately 5-12 days compared to 21 or more for agar plates), and higher sensitivity5. Disadvantages include (1) dependence on researchers having access to a suitable instrument, (2) the purchase of instrument bottles can add significantly to the cost per sample. In addition, (3) detection of growth depends on bacillary replication: sub-populations of non-replicating dormant mycobacteria will not give a signal23,24. However, this drawback also applies to growth on solid media and prolonged incubation in liquid media may restore growth of these sub-populations25,26. Furthermore, (4) using this liquid broth culture method necessitates manual harvesting of lysates and would not be suitable for medium to high throughput workflows required to screen compound libraries. In such cases, automated imaging systems are a more practical option27. However, even in high throughput experiments, it is essential to confirm the antimicrobial activity of hits using a method that measures growth directly28. In the future, the authors plan to use this method to assess the bactericidal effects of autophagy-inducing drugs in combination with conventional anti-tubercular drugs on Mtb survival in macrophages.
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 |