In vitro assessment of drug activity against Mycobacterium ulcerans

Abstract

As acknowledged by the Clinical and Laboratory Standards Institute (CLSI), there is an insufficient evidence base on which to recommend a standard method for antimicrobial susceptibility testing against M. ulcerans. The agar proportion method has been recognized as the standard method for susceptibility testing against Mycobacterium tuberculosis complex (MTBC) isolates for decades [1]. While it is more labor-intensive and requires larger amounts of drug or compound than broth-based testing, we recommend the agar proportion method for determination of minimum inhibitory concentrations against M. ulcerans. Herewith we present the method we implemented in our laboratory over the last 2 decades.

1. Introduction

Different methods are used for in vitro testing of antimicrobial agents against mycobacteria, including Mycobacterium ulcerans. As acknowledged by the Clinical and Laboratory Standards Institute (CLSI), there is an insufficient evidence base on which to recommend a standard method for antimicrobial susceptibility testing against M. ulcerans [1]. While we may use broth dilution assays to screen new compounds for activity against M. ulcerans [2], we consider the agar proportion method to be the most reliable method for testing the susceptibility of M. ulcerans to a drug or compound. The agar proportion method, which is used to determine the minimum inhibitory concentration (MIC) of a drug by comparing the growth of the bacteria on drug-containing and drug-free agar, was first used for Mycobacterium tuberculosis complex (MTBC) by Canetti et al. [3] and has been recognized as the standard method for susceptibility testing against MTBC isolates for decades [1].

This method allows relatively cost-efficient determination of the MIC of drugs and new drug candidates against M. ulcerans to identify potent agents, which can then be further tested in other models, like the mouse footpad infection model.

We define the MIC as the lowest concentration which inhibits >99% of the growth of M. ulcerans compared to growth in the absence of the drug (also see Note 6).

In the following sections, we describe in detail how to determine the MIC. The initial bacterial isolates to be tested can come from different sources, but the principle is always the same. As the described method takes at least 8 weeks to produce results, it has limited utility for the treatment of individual patients in the clinical setting. Its principal use is in laboratories to assess MIC distributions against panels of clinical isolates and to test the activity of new drug candidates.

2. Materials

All solid materials are autoclaved for 15 minutes at 121°C.

2.1. Preparation of bacterial inoculum

1. Sterile 125 ml polycarbonate Erlenmeyer flask.

2. Autoclavable 2000 ml Erlenmeyer flask.

3. 100 μl - 1000 μl pipette.

4. Autoclaved 1000 μl pipette tips.

5. Pipettor.

6. Sterile 25 ml serological pipettes.

7. Spectrometer capable of reading optical density at 600 nm (OD₆₀₀).

8. Autoclave.

9. Autoclave tray.

10. Middlebrook 7H9 broth powder.

11. Sterile Oleic acid-dextrose-catalase complex (OADC): dissolve 5 g Bovine Albumin Fraction V, 2 g Dextrose, 4 mg Catalase, 0.05 g Oleic Acid and 0.85 g Sodium Chloride in 100 ml distilled water.

12. Tween 80.

13. Glycerol.

14. M. ulcerans culture.

2.2. Preparation of drug-containing media

1. Sterile Petri dishes.

2. Autoclavable 2000 ml Erlenmeyer flask.

3. Autoclaved 400 ml beaker.

4. 100 μl - 1000 μl pipette.

5. 20 μl - 200 μl pipette.

6. Autoclaved 1000 μl and 200 μl pipette tips.

7. Pipettor.

8. Sterile 25 ml serological pipettes.

9. Autoclave.

10. Autoclave tray.

11. Sterile 2 ml Eppendorf tubes.

12. Sterile phosphate buffered saline (PBS).

13. Middlebrook 7H9 broth powder.

14. Middlebrook 7H11 agar powder.

15. OADC.

16. Glycerol.

17. Test drugs.

3. Methods

M. ulcerans is classified as an agent which should be handled under biosafety level two or three, depending on local or national regulations. The bacteria and infected clinical material should be handled in a functioning biosafety cabinet, where available. This also facilitates careful aseptic handling procedures which are necessary to prevent contamination because of the long incubation required for M. ulcerans [4].

3.1. Preparation of Middlebrook 7H9 media

1. Add 900 ml of deionized water to an autoclavable 2000 ml Erlenmeyer flask, then add 2 ml of glycerol, 0.5 g of Tween 80, and 4.7 g of Middlebrook 7H9 powder. Mix well until the powder is completely dissolved.

2. Autoclave the mixture in an autoclave tray for 10 minutes at 121°C.

3. Let the autoclaved media stand until it is cooled to ~45°C then aseptically add 100 ml of sterile OADC.

3.2. Preparation of bacterial Inoculum

1. Fill a 125 ml polycarbonate Erlenmeyer flask with 25 ml of the autoclaved OADC-enriched 7H9 broth.

2. Add to the growth media 2 ml of bacterial culture with an OD₆₀₀ between 0.6 and 1 of the M. ulcerans strain you want to test, close the Erlenmeyer flask and incubate in ambient atmosphere, without shaking at 30–32°C for 28 days.

3. At day 28 of incubation, remove 1000 μl of broth culture from the Erlenmeyer flask and measure the OD₆₀₀.

4. Continue incubating the M. ulcerans culture and repeat the OD₆₀₀ measurement (step 3) every week until the OD value is between 0.6 and 1.0.

5. When the OD₆₀₀ is between 0.6 and 1.0 dilute the broth culture to an OD₆₀₀ of 0.1 (see Notes 1 and 2).

6. The broth culture with an OD₆₀₀ of around 0.1 will be used to inoculate the drug-containing 7H11 media for determination of MIC.

3.3. Preparation of Middlebrook 7H11 media

1. Add 900 ml of deionized water to an autoclavable 2000 ml Erlenmeyer flask, then add 5 ml of glycerol and 21 g of Middlebrook 7H11 agar powder (NOTE: the exact amount might depend based on the manufacturer). Mix well until the powder is completely dissolved.

2. Autoclave the mixture for 15 minutes at 121°C.

3. Let the autoclaved agar mixture stand in a 55°C water bath until it reaches a temperature of approximately 55°C.

3.4. Determination of MIC with agar proportion method

1. Take 100 μl of the broth culture (3.2 step 6) and add it to a 2ml Eppendorf tube containing 900 μl PBS (10-fold dilution of the broth culture with an OD₆₀₀ of 0.1).

2. Repeat step 1 to produce 100-fold, 1,000-fold and 10,000-fold dilutions of the broth culture with an OD₆₀₀ of 0.1.

3. Dissolve the test drug in an appropriate solvent (see Note 3).

4. Remove the 7H11 agar from the water bath and add 100 ml sterile OADC.

5. Put 150 ml of the OADC-enriched 7H11 agar in a pre-warmed glass container (e.g., an autoclaved 400 ml beaker).

6. Pour 25 ml of the 7H11 agar in each of six petri dishes (drug-free control plates).

7. Put 100 ml of the OADC-enriched 7H11 agar in a pre-warmed glass container (e.g., an autoclaved 400 ml beaker).

8. Add to the 100 ml OADC-enriched 7H11 agar the test drug in the highest desired concentration and mix well [5] (see Note 4).

9. Pour 25 ml of the drug-containing 7H11 media into each of two petri dishes.

10. Add 50 ml of drug-free 7H11 media to the beaker containing the remaining 50 ml of drug-containing Middlebrook 7H11 agar, thereby reducing the drug concentration by 50%.

11. Repeat steps 9 and 10 until you reach the lowest concentration of drug to be tested (see Note 5).

12. Let the petri dishes stand at room temperature for one hour until the agar is solidified.

13. Plate 500 μl aliquots of the M. ulcerans broth culture in 100-fold, 1,000-fold and 10,000-fold dilutions in duplicate onto drug-free control plates.

14. Plate 500 μl aliquots of the M. ulcerans broth culture undiluted and in 100-fold dilutions in duplicate onto every concentration of the drug-containing test plates.

15. Incubate the plates for 8 weeks.

16. Read the plates. The MIC is defined as the concentration of the drug which inhibits at least 99% of bacterial growth (see Note 6).