M. ulcerans growth
M. ulcerans 1615 was obtained from Dr. Pamela Small (University of Tennessee, Knoxville, TN, USA). Bacteria were grown on Middlebrook 7H10 agar (Difco) supplemented with 10 % oleic acid-bovine serum albumin-dextrose-catalase (OADC), 0.05 % Tween 20, and 0.5 % glycerol (supplemented M7H10 agar) at 32 °C. Bacterial inocula were serially passaged through a 25-guage needle to disrupt mycobacterial clumps, diluted in PBS (pH 7.4) to obtain the desired concentration based on OD600 measurements, and quantified by plating on supplemented M7H10 agar to determine viable colony forming units (CFU).
Clay mixtures and clay mixture leachate preparation
Antibacterial, mineralogical, and chemical characteristics of the CB07, CB08, CB09, CB10, and BY07 clay mixtures and derived leachates have been previously described [18–20]. The CB clay mixtures are all composed of approximately 52 % clays and 48 % non-clay minerals . The most abundant clay mineral in all of the CB samples is illite-smectite (36–37 %), followed by montmorillonite (9.7–14.2 %) and kaolinite (1.4–3.6 %). The most abundant non-clay mineral present in the CB samples is quartz (34–37.3 %), followed by pyrite (4–5.5 %) and jarosite (2.4–4.7 %) . The most abundant minerals present in the BY07 clay mixture are Ca-smectite clay (37.3 %), followed by anorthoclase feldspar (23.0 %) and quartz (13.7 %) . All clay mixtures were sterilized by autoclaving for 1 h at 121 °C prior to experimental use. Derived leachates were obtained by vigorously stirring clay mixtures (1 g/20 ml) in sterile, deionized H2O (dH2O) for 24 h. Subsequently, the hydrated clay mixture suspensions were centrifuged (31,000 × g) for 3 h at 4 °C to separate insoluble and soluble fractions. The aqueous supernatant (leachate) was collected and sterilized by passage through a 0.22 μm filter.
In vitro antimicrobial susceptibility testing
M. ulcerans 1615 were exposed to CB07, CB08, CB09, CB10, and BY07 clay mixtures or derived leachates [18, 19], and survival was determined by enumerating colonies on supplemented M7H10 agar plates. Briefly, 100 mg of sterilized clay minerals were added to late-logarithmic phase M. ulcerans in supplemented M7H9 broth (1 ml), and the bacteria-clay suspensions were incubated at 32 °C on a rotating drum. To assess antimicrobial activity of leachates, late-logarithmic phase M. ulcerans 1615 was pelleted, resuspended in 1 ml of CB07, CB08, CB09, CB10, or BY07 clay leachates, water, or supplemented M7H9 broth, and incubated at 32 °C on a rotating drum. Experimental samples and controls were collected on 0, 7, 14, 21, and 28 days, subjected to 10-fold serial dilutions, plated on supplemented M7H10 agar, and incubated at 32 °C to determine viable CFU. Bactericidal activity is defined as the ability to kill bacteria, while bacteriostatic activity means that the agent prevents the growth of bacteria. Minimal bactericidal concentration (MBC) is defined as the lowest concentration of a particular antibacterial agent that kills ≥ 99.9 % of the bacterial population in a liquid medium.
Bacterial inoculum preparation for in vivo studies
To determine the M. ulcerans 1615 inoculum required to establish a visible ulcer, groups of four female BALB/c mice, aged four to six weeks, were injected in the tail with 103, 105, or 107 CFU of M. ulcerans in 30 μl PBS. Aliquots of M. ulcerans 1615 inoculum, grown in supplemented M7H9 broth to late-logarithmic phase of growth, were prepared as described above.
Animal infection procedures and bacterial enumeration of M. ulcerans-infected mouse tails
A total of 30 four- to-six week old female BALB/c mice (Charles River Laboratories) were subcutaneously injected in the tail with ~106
M. ulcerans 1615 cells in 30 μl of PBS. During M. ulcerans inoculation, the mice were anesthetized by intraperitoneal injection of 0.05 ml per 25 g of body weight with a mixture containing 21 mg ketamine, 2.4 mg xylazine, and 0.3 mg acepromazine. Five additional mice injected with PBS served as negative controls. On day one post inoculation, five mice infected with M. ulcerans were euthanized, and the tails were used to enumerate M. ulcerans in the infected animals. Briefly, tails were minced with blades, ground with a Potter-Elvehjem homogenizer in 0.15 M NaCl, and decontaminated with an equal volume of N-acetyl-l-cysteine sodium hydroxide. Aliquots of serial dilutions were plated on supplemented M7H10 agar, and the plates were incubated at 32 °C for 8-10 weeks. Mice were monitored daily. Of the 25 remaining animals infected with M. ulcerans, only 15 animals developed a visible ulcer. Five animals were euthanized to enumerate bacterial load before treatment, and the remaining 10 animals were either treated with hydrated clay poultices (n = 5) or left untreated (n = 5). All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Arizona State University Animal Care and Use Committee (protocols 09-1030R and 12-1240R) and conducted according to relevant national and international guidelines.
Topical application of hydrated clay poultices
Approximately three months postinfection, mice were either treated with hydrated clay at the site of infection or cleansed with sterile saline (no treatment). Mice in the clay treatment group (n = 5) were treated with hydrated CB09 clay (~0.2 grams clay mixed with 100 μl sterile dH2O) at the site of M. ulcerans infection. During topical application of hydrated clay poultices, mice were restrained using a commercial acrylic mouse restrainer. The infected area of the tail was initially washed with cotton swabs dipped in sterile dH2O. Subsequently, a sterile cotton swab was used to evenly apply a thick poultice of hydrated clay to the site of infection. Infected and treated tails were then covered with Tegaderm waterproof transparent dressings followed by an adhesive bandage to prevent the mice from disturbing and removing the clay poultice. For five consecutive days per week, the Tegaderm dressing and adhesive bandages were removed, and the infected tails were cleansed with sterile dH2O. After gentle washing to remove the clay poultice, freshly hydrated clay was applied, and the tails were redressed with bandages. Mice in the no treatment group (n = 5) did not undergo any treatment at the site of M. ulcerans infection. Similar to the infected and treated animals, the infected area of the tails of mice in the no treatment group was subjected to gentle washing with sterile water and subsequent redressing with Tegaderm and an adhesive bandage for five consecutive days per week. Decreased erythema and ulceration and reduction in bacterial load were considered indicators of wound healing. Two animals, one in the clay treatment group and one in the no treatment group, developed secondary infections and were euthanized prior to the end of the study. After 22 days of treatment, the remaining eight mice were euthanized. To enumerate bacterial load, the tails of the euthanized animals were removed aseptically, weighed, and homogenized as described above. The number of viable M. ulcerans cells in the tissue was determined by plating serial dilutions on supplemented M7H10 agar. The CFU were counted after 8-10 weeks of incubation at 32 °C and expressed as CFU/g tissue.
We used ANOVA with Tukey’s or Dunnett’s multiple comparisons to assess statistical significance. Each P value was adjusted to account for multiple comparisons of mean values across groups. Data were analyzed using GraphPad Prism 6.