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Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans , secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log 10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU. Combination therapy for Buruli ulcer (BU) is suboptimal. Here, Liu et al. show that the candidate drug TB47 has potent bactericidal activity against Mycobacterium ulcerans in vitro and in a mouse model, which underscores its potential for shortening the course of BU and treating other mycobacterial diseases.

Key Points Buruli ulcer is an emerging human disease associated with watercourses. It is especially prevalent in West African countries, such as Ghana or Benin, but also occurs in parts of Australia. Buruli ulcer results from infection with Mycobacterium ulcerans , a slow-growing toxin producer that is found in aquatic habitats. There is growing evidence to suggest that insects can act as vectors, as M. ulcerans has been found in water bugs, water striders and mosquitoes, although this is still controversial. M. ulcerans is a descendant of the ubiquitous fast-growing Mycobacterium marinum , which has undergone reductive evolution, gene decay and genome downsizing, probably after horizontal acquisition of a virulence plasmid. The virulence plasmid carries genes for huge polyketide synthases: assembly line enzymes that produce a macrolide toxin with multiple activities that is known as mycolactone. Mycolactone is cytotoxic and has immunosuppressive properties for professional antigen-presenting cells. It prevents dendritic cells from priming cellular immune responses and producing the chemotactic signals that are crucial for inflammation. Humans contract Buruli ulcer following infection with Mycobacterium ulcerans , a slow-growing toxin producer that evolved from Mycobacterium marinum . Both M. ulcerans and M. marinum are waterborne, but M. ulcerans is associated with various insects that might serve as vectors. This Review summarizes recent findings and explains how the toxin, a polyketide called mycolactone, acts on immune cells. Buruli ulcer is an emerging human disease caused by infection with a slow-growing pathogen, Mycobacterium ulcerans , that produces mycolactone, a cytotoxin with immunomodulatory properties. The disease is associated with wetlands in certain tropical countries, and evidence for a role of insects in transmission of this pathogen is growing. Comparative genomic analysis has revealed that M. ulcerans arose from Mycobacterium marinum , a ubiquitous fast-growing aquatic species, by horizontal transfer of a virulence plasmid that carries a cluster of genes for mycolactone production, followed by reductive evolution. Here, the ecology, microbiology, evolutionary genomics and immunopathology of Buruli ulcer are reviewed.

Surgical debridement was the standard treatment for Mycobacterium ulcerans infection (Buruli ulcer disease) until WHO issued provisional guidelines in 2004 recommending treatment with antimicrobial drugs (streptomycin and rifampicin) in addition to surgery. These recommendations were based on observational studies and a small pilot study with microbiological endpoints. We investigated the efficacy of two regimens of antimicrobial treatment in early-stage M ulcerans infection. In this parallel, open-label, randomised trial undertaken in two sites in Ghana, patients were eligible for enrolment if they were aged 5 years or older and had early (duration <6 months), limited (cross-sectional diameter <10 cm), M ulcerans infection confirmed by dry-reagent-based PCR. Eligible patients were randomly assigned to receive intramuscular streptomycin (15 mg/kg once daily) and oral rifampicin (10 mg/kg once daily) for 8 weeks (8-week streptomycin group; n=76) or streptomycin and rifampicin for 4 weeks followed by rifampicin and clarithromycin (7·5 mg/kg once daily), both orally, for 4 weeks (4-week streptomycin plus 4-week clarithromycin group; n=75). Randomisation was done by computer-generated minimisation for study site and type of lesion (ulceration or no ulceration). The randomly assigned allocation was sent from a central site by cell-phone text message to the study coordinator. The primary endpoint was lesion healing at 1 year after the start of treatment without lesion recurrence or extensive surgical debridement. Analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT00321178. Four patients were lost to follow-up (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, three). Since these four participants had healed lesions at their last assessment, they were included in the analysis for the primary endpoint. 73 (96%) participants in the 8-week streptomycin group and 68 (91%) in the 4-week streptomycin plus 4-week clarithromycin group had healed lesions at 1 year (odds ratio 2·49, 95% CI 0·66 to infinity; p=0·16, one-sided Fisher's exact test). No participants had lesion recurrence at 1 year. Three participants had vestibulotoxic events (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, two). One participant developed an injection abscess and two participants developed an abscess close to the initial lesion, which was incised and drained (all three participants were in the 4-week streptomycin plus 4-week clarithromycin group). Antimycobacterial treatment for M ulcerans infection is effective in early, limited disease. 4 weeks of streptomycin and rifampicin followed by 4 weeks of rifampicin and clarithromycin has similar efficacy to 8 weeks of streptomycin and rifampicin; however, the number of injections of streptomycin can be reduced by switching to oral clarithromycin after 4 weeks. European Union (EU FP6 2003-INCO-Dev2-015476) and Buruli Ulcer Groningen Foundation.

Buruli ulcer (BU) is a chronic and debilitating skin disease caused by the environmental pathogen, (MU). The primary virulence determinant is mycolactone, a cytotoxic lipid compound unique to MU and its other mycolactone producing mycobacteria (MPM) ecological variants. Although BU prevalence is highest in West Africa and Australia, little is known about MU and other MPM distribution in non-endemic regions such as the Southeastern United States (US). In this study, environmental samples (water filtrand, plant biofilm, soil, aquatic invertebrates) were collected from nine freshwater sites across Louisiana, Mississippi and Alabama over three sampling periods (August 2020, November 2020, March 2021). Samples were screened for MU and MPM presence and abundance by PCR and genotyped using variable number tandem repeat (VNTR) profiling. All nine sites were positive for MU or other MPM DNA in at least one substrate, except invertebrates. Overall, mean concentrations were 4.3 × 10 genome units (GU)/sample in August 2020, 1.26 GU/sample in November 2020, and 55.5 GU/sample in March 2021. Profiling by VNTR identified four MU (designated A-D) and one genotype(s), among environmental samples, with genotype frequencies varying by site and sampling time. Detection of MU and genotypes in Southeastern US aquatic environments, matching those from BU endemic regions, provides rationale for ongoing surveillance. Our findings broaden the known geographic range of MU and MPMs and offer baseline data to help predict and prevent and predict the possibility of zoonotic transmission in Southeastern US.

There are limited data on the epidemiology, diagnosis and optimal management of nontuberculous mycobacterial (NTM) disease in children. Retrospective cohort study of NTM cases over a 10-year-period at a tertiary referral hospital in Australia. A total of 140 children with NTM disease, including 107 with lymphadenitis and 25 with skin and soft tissue infections (SSTIs), were identified. The estimated incidence of NTM disease was 0.6-1.6 cases / 100,000 children / year; no increasing trend was observed over the study period. Temporal analyses revealed a seasonal incidence cycle around 12 months, with peaks in late winter/spring and troughs in autumn. Mycobacterium-avium-complex accounted for most cases (77.8%), followed by Mycobacterium ulcerans (14.4%) and Mycobacterium marinum (3.3%). Polymerase chain reaction testing had higher sensitivity than culture and microscopy for acid-fast bacilli (92.0%, 67.2% and 35.7%, respectively). The majority of lymphadenitis cases underwent surgical excision (97.2%); multiple recurrences in this group were less common in cases treated with clarithromycin and rifampicin compared with clarithromycin alone or no anti-mycobacterial drugs (0% versus 7.1%; OR:0.73). SSTI recurrences were also less common in cases treated with two anti-mycobacterial drugs compared with one or none (10.5% versus 33.3%; OR:0.23). There was seasonal variation in the incidence of NTM disease, analogous to recently published observations in tuberculosis, which have been linked to seasonal variation in vitamin D. Our finding that anti-mycobacterial combination therapy was associated with a reduced risk of recurrences in patients with NTM lymphadenitis or SSTI requires further confirmation in prospective trials.

Background The isolation and culture of Mycobacterium ulcerans (Mu) as a primary diagnostic modality for Buruli ulcer (BU) disease are limiting due to their low sensitivity and slow-growing nature. M. ulcerans cultures can also be overgrown with other bacteria and fungi. Culture, however, remains an important tool for the study of persisting viable M. ulcerans , drug susceptibility tests, and other molecular assays to improve management of the disease. The challenge of contamination with other fast-growing bacteria necessitates decontamination of clinical samples prior to culturing, but current methods may be too harsh, resulting in low yields of M. ulcerans . We aimed to evaluate a Tika-Kic decontamination process for M. ulcerans that uses supplements to stimulate M. ulcerans growth to improve recovery. Methods Swab and Fine Needle Aspirate (FNA) samples were collected from 21 individuals with confirmed BU at baseline (week 0) and weeks 2 and 4 after initiating antibiotic treatment. Samples were decontaminated with Tika-Kic decontamination medium and the modified Petroff (NaOH) methods then inoculated each into Mycobacterium Growth Indicator Tube (MGIT) or Löwenstein Jensen (LJ) medium. Time to growth detection and confirmation by qPCR as well as the proportion of positive cultures for all three methods and the proportion of positive cultures for all three time points were documented. Common contaminating bacteria were also isolated and identified. Results The proportion of M. ulcerans positive cultures obtained was higher for Tika-MGIT samples [14/43 (32%)] compared to Petroff-MGIT samples [10/43 (23%)] and Petroff-LJ samples [8/43 (19%)]. Baseline samples had a higher isolate proportion [17 (53%)] compared to samples collected after treatment initiation [9 (28%) for week 2 and 6 (19%) for week 4]. Contaminating bacteria isolated include Burkholderia cepacia , Pseudomonas aeruginosa , Pasteurella pneumotropica , Proteus mirabilis , Morganella morganii , Staphylococcus aureus and Enterococcus . Conclusion Our study shows an advantage for culturing Mycobacterium ulcerans from clinical samples using the Tika-Kic decontamination and growth medium. Further research is needed to refine sample processing to improve M. ulcerans recovery.

We describe emergence of Buruli ulcer in urban Geelong, Victoria, Australia, and examine timing and proximity of human cases to detection of Mycobacterium ulcerans DNA in possum feces. M. ulcerans-positive feces preceded human cases by up to 39 months, constituting an early warning of impending risk for Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc :aa . While the cytochrome bc :aa is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc :aa to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 μM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.

Chronic wounds pose a significant healthcare burden in low- and middle-income countries. Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, causes wounds with high morbidity and financial burden. Although highly endemic in West and Central Africa, the presence of BU in Sierra Leone is not well described. This study aimed to confirm or exclude BU in suspected cases of chronic wounds presenting to Masanga Hospital, Sierra Leone. Demographics, baseline clinical data, and quality of life scores were collected from patients with wounds suspected to be BU. Wound tissue samples were acquired and transported to the Swiss Tropical and Public Health Institute, Switzerland, for analysis to detect Mycobacterium ulcerans using qPCR, microscopic smear examination, and histopathology, as per World Health Organization (WHO) recommendations. Twenty-one participants with wounds suspected to be BU were enrolled over 4-weeks (Feb-March 2019). Participants were predominantly young working males (62% male, 38% female, mean 35yrs, 90% employed in an occupation or as a student) with large, single, ulcerating wounds (mean diameter 9.4cm, 86% single wound) exclusively of the lower limbs (60% foot, 40% lower leg) present for a mean 15 months. The majority reported frequent exposure to water outdoors (76%). Self-reports of over-the-counter antibiotic use prior to presentation was high (81%), as was history of trauma (38%) and surgical interventions prior to enrolment (48%). Regarding laboratory investigation, all samples were negative for BU by microscopy, histopathology, and qPCR. Histopathology analysis revealed heavy bacterial load in many of the samples. The study had excellent participant recruitment, however follow-up proved difficult. BU was not confirmed as a cause of chronic ulceration in our cohort of suspected cases, as judged by laboratory analysis according to WHO standards. This does not exclude the presence of BU in the region, and the definitive cause of these treatment-resistance chronic wounds is uncertain.