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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Methods Mol Biol. 2022;2387:195–207. doi: 10.1007/978-1-0716-1779-3_19

Drug efficacy testing in the mouse footpad model of Buruli ulcer

Paul J Converse 1, Deepak V Almeida 1, Eric L Nuermberger 1
PMCID: PMC8996816  NIHMSID: NIHMS1793057  PMID: 34643914

Abstract

Great progress has been made in understanding the pathogenesis and treatment of Buruli ulcer over the last 20 years. The rediscovery of the mouse footpad model of the disease with translation to clinical practice has changed treatment of this infectious disease, caused by Mycobacterium ulcerans, from surgery and skin grafting to the administration of antibiotics for 8 weeks or less with superior cure rates. Here we describe the development and enhancement of the mouse model during the last two decades.

Keywords: Buruli ulcer, Mycobacterium ulcerans

1. Introduction

The mouse footpad model of Mycobacterium ulcerans infection was first described by Frank Fenner (1–3) not long after the first description of what was then called Bairnsdale ulcer (4), now Buruli ulcer. After injection of a small volume of M. ulcerans broth culture, 50 μl, in mouse footpads, Fenner (2) observed a dose-dependent increase in swelling over time. Other routes of inoculation required higher concentrations of bacteria and longer times for the development of lesions. Fenner concluded that the pathogenic behavior of M. ulcerans, like its growth on culture media, is controlled by the local environmental temperature, i.e., occurring at temperatures no greater than 33°C. Measurements of footpad temperatures in nine mouse strains revealed an average of 30.00±0.45°C, approximately 5° less than the average rectal temperature (5).

Having observed that growth of M. ulcerans in vitro was not as sensitive as M. tuberculosis to para-aminosalicylic acid and was not sensitive to isoniazid or thiosemicarbazone, but was sensitive to streptomycin (STR), Leach and Fenner (3) used the mouse footpad model to show that swelling of footpads could be prevented by 1.5 mg streptomycin administered intraperitoneally daily (6 days per week) in mice treated immediately after infection and inhibited from progressing in mice with early lesions. Treatment of advanced footpad lesions yielded ambiguous results due to intercurrent infections.

Apart from the adoption of the footpad model by Charles Shepard, and later others, for studies of M. leprae (1, 6), the model was rarely used to assess susceptibility of M. ulcerans to antibiotics and extensive surgery remained the treatment of choice over the next five decades. Lunn and Rees (7) observed that clofazimine had some activity in the mouse, but not humans. Pattyn and Royackers (8) confirmed the activity of STR using the footpad model. Stanford and Phillips (9), and later Havel and Pattyn (10), showed that rifampin (RIF) had some benefit. All of these studies involved monotherapy rather than combined antibiotic treatment regimens, although Havel and Pattyn did propose that it would be rational to assess combined therapeutic regimens. However, RIF was very expensive still in the 1970s and was typically reserved for tuberculosis, and later leprosy, treatment (1).

By the 1990s, recognition of the rising number of cases of Buruli ulcer in West Africa led to a commitment by the World Health Organization to combat the disease and the establishment of the Global Buruli Ulcer Initiative in 1998. Among the many research initiatives and accomplishments that followed were those that resulted in testing of antibiotics and antibiotic combinations (11–15) in the mouse footpad model that will be described below based on our own experience and further development of the model (1, 16–24). Other sites of inoculation, e.g., the ears (25) and the tail (26), have also been utilized but the footpad model has predominated in studies evaluating antibiotic activity.

2. Materials

Use distilled water or deionized water for reagent preparation. Sterilize reagents and materials by autoclaving as indicated.

  1. Mouse strain: BALB/c mice 6 weeks of age (see note 1).

  2. Mycobacterium ulcerans strains: Strains Mu1059, Mu1615, and Mu1059 AL (see Note 2)

  3. Middlebrook 7H9 broth with 10% OADC: To make 1 liter of complete medium, dissolve 7H9 powder in 900 ml of distilled or de-ionized water, please follow manufacturer instructions amount may vary depending on manufacturer. Add 2 ml glycerol. Autoclave at 121° C for 10 minutes and cool to room temperature before aseptically adding 100 ml of Middlebrook OADC growth supplement. Store at 4° C for no more than 2 months.

  4. Middlebrook 7H11 Agar with 10% OADC, Selective: Suspend the agar powder in 900 ml distilled or de-ionized water, please follow manufacturer instructions amount may vary depending on manufacturer. Add 5 ml glycerol and swirl to make an even suspension. Autoclave at 121° C for 10 minutes and cool to 50–55° C. Aseptically add 100 ml Middlebrook OADC growth supplement, followed by the four antibiotics at total concentration as indicated carbencillin (50 mg), Polymixin B (200,000 U), trimethoprim (20 mg) and Cyclohexamide (50 mg). Pour Agar aseptically in 22–23 ml amount in standard size (100 mm × 15 mm) sterile petri dishes. Let the agar cool and solidify, turn over and make sure the plates are dry. Store in 4° C for no more than 2 months

  5. Sterile loop 10 μl

  6. Sterile 50 ml conical bottom falcon tubes or similar

  7. Luminometer: Turner systems TD 20/20 luminometer

  8. Acid fast stain kit (see note 3)

  9. Biosafety Cabinet, Class 2 or above

  10. Sterile ½ cc U-100 insulin syringe with 28G ½ inch needle

  11. Mouse restrainer: Rodent injection cone (See Note 4)

  12. Isoflurane or carbon dioxide for mouse euthanasia

  13. Sterile 70% Isopropyl alcohol swabs

  14. Sterile #10 scalpels

  15. Sterile 11.5 cm forceps

  16. Sterile 11.5 cm curved scissors

  17. Phosphate buffered saline (PBS) 1X pH 7.4: To make 1-liter solution, start with 800 ml of distilled water, add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, adjust the pH to 7.4 with HCl, add distilled water to a total volume of 1 liter. Dispense the solution into aliquots and sterilize by autoclaving (20 min, 121°C, liquid cycle). Store at room temperature.

  18. Sterile petri dishes (100 mm × 15 mm).

  19. Sterile 1 ml U-100 insulin syringe with a 25G 1-inch needle

  20. Mouse gavage needles: Both flexible or rigid needles may be used depending on user preference. The recommended size for mice is between 18–22 G.

  21. Sterile 1 ml U-100 insulin syringe

  22. Ketamine/xylazine solution: Prepare a 10 ml solution with, 1.75 ml ketamine (100 mg/mL), 0.25 ml xylazine (100 mg/ml) and 8 ml saline or sterile water for injection. 0.1 ml per 20 gm mouse weight intraperitoneally (IP), delivers a dose of 87.5 mg/kg Ketamine and 12.5 mg/kg Xylazine

  23. Glass beads (3 mm) (Sterile)

  24. Bench top Vortex

  25. Spectrophotometer capable of measuring Optical density (OD) at 600 nm

  26. Screw capped tubes, 2 ml (Sterile)

  27. Ziploc bags or other sealable bags

3. Methods

All procedures should be carried out in a biosafety cabinet, in accordance with local institutional policies for biosafety and animal care and use.

3.1. Inoculum preparation:

Our preference is to use freshly passaged bacteria contained in recently harvested footpad homogenates from swollen mouse footpads. Such isolates by definition have shown one facet of virulence (See note 5). However, if footpad homogenate is unavailable, a starter inoculum can be prepared in-vitro.

  1. For in-vitro starter inoculum, grow the required strain in 20 ml 7H9 broth with glycerol without tween. Optimum temperature for M ulcerans growth is 32° C and requires incubation time of 6–8 weeks due to its slow growth rate. Alternatively colonies can also be used from growth on solid 7H11 agar plates.

  2. After required growth is obtained, transfer the contents to a 50 ml sterile conical tube. For growth on solid media, scrape and transfer all the colonies possible using sterile loop to 50 ml tube containing 20 ml 1X PBS. Add sterile glass beads up to 5 ml mark and homogenize the culture by vortexing for 30 seconds, repeat 4–5 times or till relatively homogenous suspension is obtained. Allow it to stand for 2–3 minutes so that the large particles settle, transfer the supernatant to a fresh tube and adjust the OD600 between 0.8–1. At this step acid fast staining of an aliquot of the inoculum is recommended to verify the purity of the in-vitro culture.

  3. Inoculate the mouse footpads as described in next section. The resultant swollen footpads should be harvested and used for subsequent infections for drug experiments.

  4. When using freshly harvested footpads, an approximate estimation of inoculum can be made using acid fast staining. Best practice is to use footpads within a couple of days since the organisms start to lose viability on long term storage, We typically aim for an inoculum of 106/ml or ~3.2×104 or (4.5log10) CFU/0.03 ml injected in the footpad. The inoculum count can be verified by plating serial ten-fold dilutions on Middlebrook 7H11 selective plates.

3.2. Footpad inoculation

  1. Use ½ cc U-100 insulin syringe with 28G ½ inch needle, the syringe must be primed, i.e., gently pumped, before use. Working aseptically, in the biosafety cabinet, carefully fill the syringe with the inoculum and make sure to remove any air bubbles. One footpad requires 0.03 ml of inoculum. Based on one’s preference either load multiple doses or a single dose in the syringe.

  2. One may infect one or both hind footpads based on the requirements and institutional policies for conducting animal experiments. Ideally two people are needed for the inoculation, one to restrain the mouse while the second person inoculates the footpad. Alternatively, if working alone, a mouse restrainer will need to be used (see note 4).

  3. Ensuring the mouse is properly restrained, grasp the footpad just above the toes using a pair of flat head forceps or fingertips (see Fig. 1) using the non-dominant hand. Ensure that the ventral side of the foot pad (imagine the palm of a human hand) faces upwards. Insert the needle with the bevel side up just under the skin between the walking pad and heel. Deliver the inoculum and retract the needle slowly, release the foot and return the mouse to the cage.

Fig. 1.

Fig. 1

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Mouse footpad inoculation. While the mouse is restrained by a partner, the footpad is grasped by the non-dominant hand and the needle is inserted just under the skin. The inoculum is delivered slowly and the needle is then retracted slowly.

3.3. Assessment of footpad swelling

Depending on the inoculum, footpads will begin to swell 4 to 6 weeks after infection. We use the Average Lesion Index scoring system developed by Grosset and colleagues (12). There are 4 swelling grades plus the normal footpad, which is Grade 0 (Fig. 2). Before reaching Grade 1, non-inflammatory footpad swelling, there are portents or changes (slight swelling) in footpad appearance approximately one week before, indicating that the process is about to reach grade 1. Similarly, the evolution can be assessed as the footpad progresses from grade 1 to Grade 2, or inflammatory footpad swelling, to Grade 3, inflammatory foot swelling, and finally to Grade 4, inflammatory leg swelling. After Grade 3, signs of ulceration may appear with cage bedding adhering to the footpad. Depending on the institutional animal care and use policies, mice should be sacrificed or treated no later than the Grade 3 stage. It should be emphasized that in spite of the swelling, mice remain fully mobile and apparently pain free due to the anesthetizing activity of the mycolactone toxin. We do our inspection and assessment visually. Others (27) have used calipers, for example, to measure footpad size but that is time consuming and does not take into account redness and the condition of the entire footpad

Fig. 2.

Fig. 2

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Footpad swelling. After a period of weeks after infection, swelling begins to be manifested until the entire upper portion (arrow) appears inflated as shown for Grade 1. Over the following one to two weeks, the swollen area will start to redden and become Grade 2. Over the subsequent one to two weeks the redness and swelling will extend to the ankle (arrow) and become Grade 3. Beyond this time, the footpad skin begins to tear making small ulcers leading to the adhesion of cage bedding material to the foot (not shown). At this point, mice are at risk of bacterial superinfection, sufficient cleansing of the foot is unreliable for obtaining cultivable bacilli and mouse locomotion is reduced. Such mice should be sacrificed for humane reasons.

3.4. Footpad Harvest

Footpads are harvested for implantation on the day after infection, at the beginning of treatment to assess the bacterial burden, and at different time points as indicated by the protocol after treatment initiation.

  1. Mice are killed by an approved method of euthanasia, such as cervical dislocation after intentional overdose of isoflurane or carbon dioxide inhalation.

  2. The footpads are carefully cleaned and disinfected by swabbing entire footpad with soap followed by sterile water and then swabbing with sterile Isopropyl alcohol swabs.

  3. Before harvesting ensure that the footpad is fully dry. Using sterile scalpel and forceps, holding the toes, make a slight nick in the tissue just distal to the ankle bone and then, slightly turning the scalpel to be parallel with the foot bones, cut along the edge of the foot bones and peel the footpad from the bone. Repeat tissue removal on the dorsal side. The tissue is then minced in a few drops of 1x PBS in a sterile petri dish using a pair of sterile curved scissors until it reaches a consistency like mashed potatoes.

  4. Transfer the minced tissue in sterile 2-ml screw capped tubes containing 1.5 ml 1x PBS, using the same scissors and disposable transfer pipettes.

3.5. Drug treatment

  1. Drug dosing: The dose or dose range of a drug or compound to be administered depends on the experimental objectives. For drugs for which human plasma exposures are known, it is typically desirable to select dosing regimens that produce similar exposures in mice, if feasible. Investigators may use historical references to establish these dosing regimens. However, it is always advisable, when feasible, to establish and validate the anticipated drug exposures in their own laboratory when using a drug for the very first time or in a new drug combination if the potential drug-drug interaction exists. This is because the drug exposures may be influenced by variables like the manufacturer, storage conditions, preparation techniques, the specific dosing formulation used, etc. It is ideal to establish the drug pharmacokinetics (PK) in infected mice but, in the case of M. ulcerans infection in mice, there is limited systemic illness and so the PK are less likely to be affected. A more detailed discussion of integrating PK into experiments in the mouse footpad model is beyond the scope of this chapter to cover the entire discussion of PK and readers are advised to refer to internet resources and the following references (28, 29) for details. It is important to establishing the single dose and steady state pk of study drugs to guide the selection of the drug dose to be used in study.

  2. Drug administration procedures: Depending on the drug’s oral bioavailability, we prefer to administer the drug by parenteral injection (e.g., subcutaneous or intraperitoneal) or oral gavage (see note 6).
    1. Subcutaneous injection. We use 1 ml U-100 insulin syringe with a 25G 1-inch needle. The volume of injection is typically 0.2 ml. Subcutaneous administration of material often causes minimal pain or discomfort, provided the material is non-irritant, has a near-neutral pH, and is largely isotonic. Ideally the drug formulation will be sterile. The two most common sites for injection are (i) the scruff of the neck and (ii) the loose skin over the flank. It may be best to switch sites of injection regularly when repeated doses are needed. It is not usually necessary to try to sterilize the skin with antiseptics – their use is almost always ineffective, and they simply prolong the duration of restraint needed and cause unnecessary stress to the animal. Ensuring that the injecting fluid is at body temperature will further reduce the discomfort felt by the mice.
    2. Oral gavage. The needle used should be based on the experience and familiarity of the person executing the procedure. We use a 20G curved needle with 1 ml U-100 insulin syringe. The amount of drug delivered is generally 1% of the body weight (e.g. for a 20-gm mouse it will be 0.2 ml). Due to the small size of stomach it is best to limit the volume and number of gavages. If giving multiple drugs it is best to co-administer them in a single gavage. However, to avoid drug-drug interactions, some drugs need to be administered individually; in this case it is best to minimize the number of gavages per day as much as possible. It cannot be emphasized enough the familiarity of the person conducting the gavages. The gavaging needle needs to be inserted gently to avoid trauma to the aerodigestive tract while being inserted deep enough to avoid aspiration of the formulation into the lungs. Animals should be observed for at least 15 mins after oral gavage for signs of distress and complications.

3.6. Assessment of treatment response

  1. Footpad swelling. In response to effective treatment, footpad swelling should decrease, reversing the swelling process outlined above. In some cases, the swelling resolves to a normal appearance. In other cases, especially when treatment is initiated with extensive footpad swelling, there is residual alteration to footpad morphology, presumably due to incomplete tissue remodeling.

  2. Measurement of relative light units (RLU). Use of an autoluminescent strain of M. ulcerans offers advantages of observation of real-time assessments of bacterial multiplication and the microbiological response to treatment by monitoring the footpad RLU counts.
    1. Intact footpad. To measure RLU in an intact footpad, individual mice must first be anesthetized with ketamine/xylazine (87.5/12.5 mg/kg, injected intraperitoneally). Then the mouse footpad is placed in a tabletop luminometer (TD 20/20) with care taken so the footpad being monitored rests fully in the well of the luminometer. Ensure that the lid of the instrument is fully closed. The RLU is then measured for 4 seconds. Other imaging systems, such as an IVIS camera, may also be used, if available.
    2. Footpad homogenate. To measure RLU in a footpad homogenate after footpad harvest, the sample simply needs to be placed in an appropriate sterile (if culture of the sample is also planned) vessel for the luminometer. The RLU are then measured for 4 seconds.

  3. Quantitative culture of footpad homogenates. We prefer to use Middlebrook 7H11 selective agar medium, however, LJ slants may also be used (see note 7). Depending on the expected CFU count range, serial tenfold dilutions of the footpad homogenates in 1x PBS are prepared and 500 μl of each dilution is plated on a 7H11 selective plate (e.g., if you expect 104 CFU/ footpad, then plating undiluted, 10−1 and 10−2 dilutions is recommended) (see note 6). Plates are then sealed in Ziploc or other sealable bags and incubated upright at 32°C, a CO2 incubator is not needed. The plates can be turned over after a couple of weeks when the inoculum is fully absorbed, however this step is not necessary. Preliminary counts are made after 6 weeks and counts are finalized after 8 weeks of incubation. During the first week of incubation, the media should be observed for any signs of contamination.

3.7. Determination of drug efficacy in mice

There are two methods that have been used to assess drug efficacy against M. ulcerans in mice. The first, adapted from the mouse footpad model of M. leprae, is termed kinetic or preventative. After inoculation of bacteria into footpads, the infection is permitted to be established over a matter of days up to no more than two weeks before the administration of drugs for a pre-determined rhythm and duration. If footpad swelling is prevented or limited or no multiplication of bacilli is observed, then the drug, or drug combination, is deemed to be active. This method remains helpful for a more rapid and less costly evaluation of new drugs alone or in combination and in dose-ranging studies, especially if combined with RLU assessments. One limitation of this approach is that it does not mimic the established infection and disease found in clinical situations. Unlike M. leprae, which classically, at least, required about 6 months to show multiplication in the mouse and cannot be cultivated in vitro, which is necessary for performing a minimum inhibitory concentration test (MIC test), growth of M. ulcerans can be detected in shorter periods of time. Curative treatment of mice infected with M. leprae would require most of the lifespan of a mouse. In contrast, drug activity can be assessed in M. ulcerans-infected mice well before they mice reach middle age. We, and others, (30, 31), therefore, adopted a curative model for assessing drug efficacy. After the establishment of swelling (e.g., grade 1) or even advanced swelling (grade 2 or even 3), treatment is initiated for a period typically of 2, 4, 6, or 8 weeks. The need for prompt initiation of therapy increases as the degree of swelling increases in order to avoid ulceration of the footpad and bacterial superinfection. Drugs or drug combinations can then be evaluated for their activity after the completion of treatment for these times by sacrificing the mice and enumerating the footpad bacillary burden. It is also possible to delay mouse sacrifice after treatment completion for 12 to 24 weeks to determine if the treatment has actually sterilized the footpads or if there has been re-growth of bacteria (i.e., relapse). However, it bears stating that the clinical relevance of relapse as an endpoint in the mouse footpad model is debatable because, unlike tuberculosis in which relapse after treatment is a significant threat, relapse of Buruli ulcer appears to be quite uncommon in patients treated with WHO-recommended drug regimens. Perhaps, as treatment succeeds in eliminating mycolactone production and restoring local host immune responses, these mechanisms are capable of preventing regrowth of the organisms. Thus, relapse may not occur in antibiotic-treated Buruli ulcer patients, whereas in tuberculosis, there may be more virulence factors and more relapse.

Notes:

  1. C57BL/6 mice may be more susceptible to M. ulcerans than BALB/c (19, 32) and are not as easy to handle. Outbred mice have greater variability in response to infection with M. ulcerans (J. Grosset, personal communication). In limited studies, we found that nude mice show greater susceptibility to progressive and disseminated disease to the entire leg (unpublished observations). A pilot experiment with C3HeB/FeJ mice showed no greater susceptibility to M. ulcerans. Others have used the FVB/N mouse strain that develops and then spontaneously heals ulcers (26).

  2. Our first experiments were with the Cu001 strain that, in our hands, appeared to have lacked virulence, though Ji et al. used it successfully in multiple chemotherapy studies (30, 31). We observed footpad swelling and multiplication of M. ulcerans strain Mu1617 (TMC 1617, ATCC 19423), the type strain first reported from Australia (4), in most but not all mouse footpads. However, we subsequently learned that this strain, presumably due to improper handling, has lost the ability to produce the virulence toxin, mycolactone. We then obtained strain Mu1615 (33), originally isolated from a Malaysian patient, and Mu1059, isolated in Ghana (34). Footpads infected with these strains all developed swelling, showed bacterial multiplication, and produced mycolactone (19). Mu1059 was successfully transformed to be autoluminescent, allowing for non-invasive, real-time assessments of bacterial replication and antimicrobial drug activity (35, 36).

  3. We use commercially available Kinyoun stain kits, however stains can be made in house provided adequate quality controls are used. Alternatively, Ziehl-Neelsen’s method can also be used.

  4. There are number of mice restrainers available, however if you plan to do footpad inoculation, the conical ones indicated work best since it allows one to slide the footpad out while immobilizing the mice.

  5. It is unclear if virulence is necessarily diminished by culture in vitro but experience in M. tuberculosis suggests this should be avoided due to changes in the cell wall that result in altered production of surface molecules, such as phthiocerol dimycoceroserates (PDIMs). Frozen specimens documented to have been obtained from a footpad lesion can be a substitute, but we have found that the time to footpad swelling can be extended by at least two weeks when using strains stored in this manner.

  6. Other possible routes include topical or inhalational administration, but these will not be discussed here. Gavage tends to be quicker in experienced hands and carries a lower risk of infection or injury than injection. The dose administered may be just as accurate with gavage as with injection, although the plasma drug exposure may be more variable with gavage due to variables that impact oral bioavailability. Still, dosing by gavage is considerably more reliable than dosing through the diet or drinking water, as food and water consumption is likely to vary between mice and day-to-day in the same mice.

  7. We prefer use of 7H11 selective media since contamination, if it occurs, tends to be isolated; alternatively, LJ slants supplemented with antibiotics can also be used. The recommended amount to be inoculated is 100 μl per slant. LJ slants need to be incubated in special angled racks so that the slant is horizontal with the caps loosened. After about one week, when the inoculum is fully absorbed on the slant, the caps may be tightened, and the slants may be incubated vertically to save incubator space.

Acknowledgements

This work has been supported by NIH grants R01-AI-082612 to Jacques Grosset and R01-AI-113266 to E.L.N. and by the Johns Hopkins Center for Tuberculosis Research.

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