A lesion in two: Buruli ulcer and squamous cell carcinoma coexistence.

The concurrent diagnoses of Buruli ulcer (BU) and cutaneous squamous cell carcinoma (SCC) is a phenomenon not previously described, despite the fact that both conditions are highly prevalent in Australia. This report presents an intriguing case of concurrent diagnoses, with clues alluding to more than one skin condition being present. The case involves a 73-year-old man with BU diagnosed on the scalp, an atypical location, which led to the consideration of malignancy, ultimately revealing concurrent SCC. This case highlights the importance of considering both conditions in patients with epidemiological risk factors, necessitating multiple lines of investigation for accurate diagnosis. Medical practitioners must remain vigilant and incorporate this possibility into their diagnostic algorithms for suspicious skin lesions to optimize treatment and outcomes. This is the first recorded instance of simultaneous diagnosis, underlining the need for enhanced awareness and attention to these unique cases.

Type-I interferons promote innate immune tolerance in macrophages exposed to Mycobacterium ulcerans vesicles.

Buruli ulcer is a chronic infectious disease caused by Mycobacterium ulcerans. The pathogen persistence in host skin is associated with the development of ulcerative and necrotic lesions leading to permanent disabilities in most patients. However, few of diagnosed cases are thought to resolve through an unknown self-healing process. Using in vitro and in vivo mouse models and M. ulcerans purified vesicles and mycolactone, we showed that the development of an innate immune tolerance was only specific to macrophages from mice able to heal spontaneously. This tolerance mechanism depends on a type I interferon response and can be induced by interferon beta. A type I interferon signature was further detected during in vivo infection in mice as well as in skin samples from patients under antibiotics regiment. Our results indicate that type I interferon-related genes expressed in macrophages may promote tolerance and healing during infection with skin damaging pathogen.

From Bacterial Toxin to Therapeutic Agent: The Unexpected Fate of Mycolactone.

"Recognizing a surprising fact is the first step towards discovery." This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a "surprising" lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells. We report in this review the main discoveries that marked our research into mycolactone, and the medical perspectives they opened up. The story of mycolactone is not over and the applications of Sec61 inhibition may go well beyond immunomodulation, viral infections, and oncology.

Changes in Inflammatory Markers in Patients Treated for Buruli Ulcer and Their Ability to Predict Paradoxical Reactions.

Mycobacterium ulcerans causes Buruli ulcer, the third most frequent mycobacterial disease after tuberculosis and leprosy. Transient clinical deteriorations, known as paradoxical reactions (PRs), occur in some patients during or after antibiotic treatment. We investigated the clinical and biological features of PRs in a prospective cohort of 41 patients with Buruli ulcer from Benin. Neutrophil counts decreased from baseline to day 90, and interleukin 6 (IL-6), granulocyte colony-stimulating factor, and vascular endothelial growth factor were the cytokines displaying a significant monthly decrease relative to baseline. PRs occurred in 10 (24%) patients. The baseline biological and clinical characteristics of the patients presenting with PRs did not differ significantly from those of the other patients. However, the patients with PRs had significantly higher IL-6 and tumor necrosis factor alpha (TNF-α) concentrations on days 30, 60, and 90 after the start of antibiotic treatment. The absence of a decrease in IL-6 and TNF-α levels during treatment should alert clinicians to the possibility of PR onset.

An Overview of 10 Years of Activity of a Molecular Laboratory for Buruli Ulcer Diagnosis at a Field Hospital in Benin.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates.

Mycobacterium ulcerans culture results according to duration of prior antibiotic treatment: A cohort study.

Mycobacterium ulcerans disease is a necrotising disease of the skin and subcutaneous tissue and is effectively treated with eight-weeks antibiotic therapy. Significant toxicities, however, are experienced under this prolonged regimen. Here, we investigated the length of antibiotic duration required to achieve negative cultures of M. ulcerans disease lesions and evaluated the influence of patient characteristics on this outcome. M. ulcerans cases from an observational cohort that underwent antibiotic treatment prior to surgery and had post-excision culture assessment at Barwon Health, Victoria, from May 25 1998 to June 30 2019, were included. Antibiotic duration before surgery was grouped as <2 weeks, ≥2-<4 weeks, ≥4-<6 weeks, ≥6-<8 weeks, ≥8-<10 weeks and ≥10-20 weeks. Cox regression analyses were performed to assess the association between variables and culture positive results. Ninety-two patients fitted the inclusion criteria. The median age was 60 years (IQR 28-74.5) and 51 (55.4%) were male. Rifampicin-based regimens were predominantly used in combination with clarithromycin (47.8%) and ciprofloxacin (46.7%), and the median duration of antibiotic treatment before surgery was 23 days (IQR, 8.0-45.5). There were no culture positive results after 19 days of antibiotic treatment and there was a significant association between antibiotic duration before surgery and a culture positive outcome (p<0.001). The World Health Organisation category of the lesion and the antibiotic regimen used had no association with the culture outcome. Antibiotics appear to be effective at achieving negative cultures of M. ulcerans disease lesions in less than the currently recommended eight-week duration.

Buruli ulcer caused by Mycobacterium ulcerans subsp. shinshuense: A case report.

Buruli ulcer is the third most common mycobacterial infection worldwide and is mainly diagnosed in tropical regions. Globally, this progressive disease is caused by Mycobacterium ulcerans; however, Mycobacterium ulcerans subsp. shinshuense, an Asian variant, has been exclusively identified in Japan. Because of insufficient clinical cases, the clinical features of M. ulcerans subsp. shinshuense-associated Buruli ulcer remain unclear. A 70-year-old Japanese woman presented with erythema on her left backhand. The skin lesion deteriorated without an apparent etiology of inflammation, and she was referred to our hospital 3 months after disease onset. A biopsy specimen was incubated in 2% Ogawa medium at 30 °C. After 66 days, we detected small yellow-pigmented colonies, suggesting scotochromogens. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI Biotyper; Bruker Daltonics, Billerica, MA, USA) indicated that the organism was Mycobacterium pseudoshottsii or Mycobacterium marinum. However, additional PCR testing for the insertion sequence 2404 (IS2404) was positive, suggesting that the pathogen was either M. ulcerans or M. ulcerans subsp. shinshuense. Further examination by 16S rRNA sequencing analysis, focusing on nucleotide positions 492, 1247, 1288, and 1449-1451, we finally identified the organism as M. ulcerans subsp. shinshuense. The patient was successfully treated with 12 weeks of clarithromycin and levofloxacin treatment. Mass spectrometry is the latest microbial diagnostic method; however, it cannot be used to identify M. ulcerans subsp. shinshuense. To accurately detect this enigmatic pathogen and uncover its epidemiology and clinical characteristics in Japan, more accumulation of clinical cases with accurate identification of the causative pathogen is essential.

Vaccination efforts for Buruli ulcer.

BACKGROUND: Buruli ulcer is one of the most common mycobacterial diseases usually affecting poorer populations in tropical and subtropical environments. This disease, caused by M. ulcerans infection, has devastating effects for patients, with significant health and economic burden. Antibiotics are often used to treat affected individuals, but in most cases, surgery is necessary. AREA COVERED: We present progress on Buruli ulcer vaccines and identify knowledge gaps in this neglected tropical disease. EXPERT OPINION: The lack of appropriate infrastructure in endemic areas, as well as the severity of symptoms and lack of noninvasive treatment options, highlights the need for an effective vaccine to combat this disease. In terms of humoral immunity, it is vital to consider its significance and the magnitude to which it inhibits or slows down the progression of the disease. Only by answering these key questions will it be possible to tailor more appropriate vaccination and preventative provisions.

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin.

BACKGROUND: Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. METHODS: We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. DISCUSSION: If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). TRIAL REGISTRATION: ClinicalTrials.gov NCT05169554 . Registered on 27 December 2021.

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity.

The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3-/-/perk-/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A1; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

Drug Efficacy Testing in the Mouse Footpad Model of Buruli Ulcer.

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.

Determination of Bioenergetic Parameters in Mycobacterium ulcerans.

The oxidative phosphorylation (OxPhos) pathway has emerged as an attractive pathway for the development of anti-mycobacterial drugs. The OxPhos pathway is essential for ATP resynthesis and maintenance of the electrochemical transmembrane gradient. The bioenergetic parameters of the pathway such as oxygen consumption rate and ATP levels are quantifiable using current technology. Measuring these parameters are useful tools to gauge rapidly the impact of drug candidates on their capacity to inhibit the OxPhos pathway in Mycobacterium ulcerans.

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.

Reconstructive surgery for sequellae of Mycobacterium ulcerans infection (Buruli ulcer) of the upper limb.

INTRODUCTION: Infection by Mycobacterium ulcerans constitutes a neglected tropical disease whose prevalence seems to have overrun those of cutaneous tuberculosis and leprosy. Its aggressivity depends on a mycolactone toxin. Lesions may involve skin, tendon and bone with a large spectrum of manifestations: non-ulcerative (papules, nodules, plaques), ulcerative and oedematous presentations as well as osteomyelitis with muscular contraction and ankylosis. Upper limbs account for more than two thirds of the infection sites. Surgical treatment may involve tendon transpositions, partial and total skin grafts. Amputation is relegated to extreme cases. MATERIAL AND METHODS: Selected iconography from patients during the last 15 years is presented. At least 1500 cases had partial skin grafts (anterior thigh). Total skin grafts (inguinal region) were used in about 200 cases. Complex lesions involved 9 ilioinguinal flaps (5 boys, 4 girls, mean age 11.2 years, range 2-16 years), 5 tendon transfers (4 boys, one girl, mean age 15.4 years, range 12-19 years) and 3 resections of the first carpal row (2 girls, 1 boy, mean age 8 years, range 4-15 years). RESULTS AND DISCUSSION: Out of 9 ilioinguinal flaps mild, marginal necrosis was the only complication in 2 patients without flap loss. Mean hospital stay was 26.44 days (range, 18-41 days), with return to full weight-bearing after a mean of 12 weeks (range 9-25 weeks) after discharge. Functional thumb opposition to allow pencil prehension was achieved in all three cases of resection of first carpal row resection without postoperative complications.

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin.

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.

The One That Got Away: How Macrophage-Derived IL-1ß Escapes the Mycolactone-Dependent Sec61 Blockade in Buruli Ulcer.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1ß was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the "second signal" for IL-1ß production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone's inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1ß function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1ß in BU pathogenesis, treatment and wound healing.

Novel Diagnostics for Kaposi Sarcoma and Other Skin Diseases in Resource-Limited Settings.

In resource-limited settings, point-of-care diagnostic devices have the potential to reduce diagnostic delays and improve epidemiologic surveillance of dermatologic conditions. We outline novel-point-of care diagnostics that have recently been developed for dermatologic conditions that primarily affect patients living in resource-limited settings, namely, Kaposi sarcoma, cutaneous leishmaniasis, leprosy, Buruli ulcer, yaws, onchocerciasis, and lymphatic filariasis. All of the technologies described in this article are prototypes, and some have undergone field testing. These devices still require validation in real-world settings and effective pricing to have a major impact on dermatologic care in resource-limited settings.

Screening anti-infectious molecules against Mycobacterium ulcerans: A step towards decontaminating environmental specimens.

Mycobacterium ulcerans, a non-tuberculous mycobacterium responsible for Buruli ulcer, resides in poorly defined environmental niches in the vicinity of stagnant water. Very few isolates have been confirmed. With a view to culturing M. ulcerans from such contaminated environmental specimens, we tested the in vitro susceptibility of the M. ulcerans CU001 strain co-cultivated with XTC cells to anti-infectious molecules registered in the French pharmacopoeia. We used a standardised concentration to identify molecules that were inactive against M. ulcerans and which could be incorporated into a decontaminating solution. Of 116 tested molecules, 64 (55.1%) molecules were ineffective against M. ulcerans CU001. These included 34 (29.3%) antibiotics, 14 (12%) antivirals, eight (6.8%) antiparasitics, and eight (6.8%) antifungals. This left 52 molecules which were active against M. ulcerans CU001. Three of the inactive antimicrobial molecules (oxytetracycline, polymyxin E and voriconazole) were then selected to prepare a decontamination solution which was shown to respect M. ulcerans CU001 viability. These three antimicrobials could be incorporated into a decontamination solution to potentially isolate and culture M. ulcerans from environmental samples.

The diversity of mycolactone-producing mycobacteria.

Mycolactone-producing mycobacteria (MPM) form an intriguing group of environmental opportunistic pathogens of mammals and human patients in whom they cause cutaneous and subcutaneous ulcers known as "Buruli ulcer" when they occur in humans. We reviewed whole genome sequence data and ecological and phenotypic characteristics from 44 MPMs and closely related Mycobacterium marinum. This analysis indicated that all the 24 M. marinum isolates were delineated into seven taxa and our comprehensive, polyphasic taxonomic approach led to the proposal of delineating M. marinum genomospecies, 01-07. Likewise, 20 MPMs isolates were delineated into seven additional M. ulcerans genomospecies, 01-07. A taxonomic card explaining the ecology, hosts of isolation and the plasmid harboured is provided for each taxon.

Risk Factors Associated with Antibiotic Treatment Failure of Buruli Ulcer.

Combination antibiotic therapy is highly effective in curing Buruli ulcer (BU) caused by Mycobacterium ulcerans Treatment failures have been uncommonly reported with the recommended 56 days of antibiotics, and little is known about risk factors for treatment failure. We analyzed treatment failures among BU patients treated with ≥56 days of antibiotics from a prospective observational cohort at Barwon Health, Victoria, from 1 January 1998 to 31 December 2018. Treatment failure was defined as culture-positive recurrence within 12 months of commencing antibiotics under the following conditions: (i) following failure to heal the initial lesion or (ii) a new lesion developing at the original or at a new site. A total of 430 patients received ≥56 days of antibiotic therapy, with a median duration of 56 days (interquartile range [IQR], 56 to 80). Seven (1.6%) patients experienced treatment failure. For six adult patients experiencing treatment failure, all were male, weighed >90 kg, did not have surgery, and received combination rifampin-clarithromycin (median rifampin dose, 5.6 mg per kg of body weight per day; median clarithromycin dose, 8.1 mg/kg/day). When compared to those who did not fail treatment on univariate analysis, treatment failure was significantly associated with a weight of >90 kg (P < 0.001), male gender (P = 0.02), immune suppression (P = 0.04), and a first-line regimen of rifampin-clarithromycin compared to a regimen of rifampin-fluoroquinolone (P = 0.05). There is a low rate of treatment failure in Australian BU patients treated with rifampin-based oral combination antibiotic therapy. Our study raises the possibility that treatment failure risk may be increased in males, those with a body weight of >90 kg, those with immune suppression, and those taking rifampin-clarithromycin antibiotic regimens, but future pharmacokinetic and pharmacodynamics studies are required to determine the validity of these hypotheses.