Recombinant Antibodies against Mycolactone

In the past, it has proved challenging to generate antibodies against mycolactone, the primary lipidic toxin A of Mycobacterium ulcerans causing Buruli ulcer, due to its immunosuppressive properties. Here we show that in vitro display, comprising both phage and yeast display, can be used to select antibodies recognizing mycolactone from a large human naïve phage antibody library. Ten different antibodies were isolated, and hundreds more identified by next generation sequencing. These results indicate the value of in vitro display methods to generate antibodies against difficult antigenic targets such as toxins, which cannot be used for immunization unless inactivated by structural modification. The possibility to easily generate anti-mycolactone antibodies is an exciting prospect for the development of rapid and simple diagnostic/detection methods.

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Environmental Variations in Mycobacterium ulcerans Transcriptome: Absence of Mycolactone Expression in Suboptimal Environments

Toxins ◽

10.3390/toxins11030146 ◽

2019 ◽

Vol 11 (3) ◽

pp. 146

Author(s):

Daniel Sanhueza ◽

Jean-François Guégan ◽

Heather Jordan ◽

Christine Chevillon

Keyword(s):

Culture Medium ◽

Expression Profiles ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Virulence Plasmid ◽

Genomic Expression ◽

Environmental Variations ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Buruli ulcer is a neglected tropical infectious disease, produced by the environmentally persistent pathogen Mycobacterium ulcerans (MU). Neither the ecological niche nor the exact mode of transmission of MU are completely elucidated. However, some environmental factors, such as the concentration in chitin and pH values, were reported to promote MU growth in vitro. We pursued this research using next generation sequencing (NGS) and mRNA sequencing to investigate potential changes in MU genomic expression profiles across in vitro environmental conditions known to be suitable for MU growth. Supplementing the growth culture medium in either chitin alone, calcium alone, or in both chitin and calcium significantly impacted the MU transcriptome and thus several metabolic pathways, such as, for instance, those involved in DNA synthesis or cell wall production. By contrast, some genes carried by the virulence plasmid and necessary for the production of the mycolactone toxin were expressed neither in control nor in any modified environments. We hypothesized that these genes are only expressed in stressful conditions. Our results describe important environmental determinants playing a role in the pathogenicity of MU, helping the understanding of its complex natural life cycle and encouraging further research using genomic approaches.

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Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening

10.1101/439828 ◽

2018 ◽

Author(s):

María Pilar Arenaz Callao ◽

Rubén González del Río ◽

Ainhoa Lucía Quintana ◽

Charles J. Thompson ◽

Alfonso Mendoza-Losana ◽

...

Keyword(s):

Inhibitory Concentration ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Beta Lactam ◽

Beta Lactams ◽

Amoxicillin Clavulanate ◽

Low Toxicity ◽

Potential Use ◽

Ulcer Treatment

ABSTRACTThe potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

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First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

10.21203/rs.2.17920/v1 ◽

2019 ◽

Author(s):

Tchalare Kondi Makagni ◽

Maman Issaka ◽

Piten Ebekalisai ◽

Disse Kodjo ◽

Essossimna A. Kadanga ◽

...

Keyword(s):

Fine Needle Aspiration ◽

Slow Growth ◽

Buruli Ulcer ◽

Needle Aspiration ◽

Mycobacterium Ulcerans ◽

Clinical Samples ◽

Direct Examination ◽

Fine Needle ◽

Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

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In Vitro Killing of Mycobacterium ulcerans by Acidified Nitrite

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.8.3130-3132.2004 ◽

2004 ◽

Vol 48 (8) ◽

pp. 3130-3132 ◽

Cited By ~ 30

Author(s):

R. Phillips ◽

S. Kuijper ◽

N. Benjamin ◽

M. Wansbrough-Jones ◽

M. Wilks ◽

...

Keyword(s):

Buruli Ulcer ◽

Mycobacterium Ulcerans

ABSTRACT Mycobacterium ulcerans, which causes Buruli ulcer, was exposed to acidified nitrite or to acid alone for 10 or 20 min. Killing was rapid, and viable counts were reduced below detectable limits within 10 min of exposure to 40 mM acidified nitrite. M. ulcerans is highly susceptible to acidified nitrite in vitro.

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Molecular characterization of Mycobacterium ulcerans DNA gyrase and identification of mutations reduced susceptibility against quinolones in vitro

10.1101/2021.09.29.462499 ◽

2021 ◽

Author(s):

Hyun Kim ◽

Shigtarou Mori ◽

Tsuyoshi Kenri ◽

Yasuhiko Suzuki

Keyword(s):

Buruli Ulcer ◽

Dna Gyrase ◽

Resistance Mechanisms ◽

Docking Studies ◽

Mycobacterium Ulcerans ◽

Amino Acid Residues ◽

Wild Type Enzyme ◽

Ulcer Disease

ABSTRACTBuruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans (Mul). Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase; however, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in Mul remains unknown. This study investigated the detailed molecular mechanism of Mul DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the Mul subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC50 of FQs against Ala91Vla and Asp95Gly mutants of Mul shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type enzyme. Similarly, the CC25 was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of WT/mutant Mul DNA gyrase and FQ side chains was assessed via molecular docking studies. This is the first detailed study showing the contribution of Mul DNA GyrA subunit mutations to reduce the susceptibility against FQs.

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Shortening Buruli Ulcer Treatment with Combination Therapy Targeting the Respiratory Chain and Exploiting Mycobacterium ulcerans Gene Decay

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00426-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 16

Author(s):

Paul J. Converse ◽

Deepak V. Almeida ◽

Sandeep Tyagi ◽

Jian Xu ◽

Eric L. Nuermberger

Keyword(s):

Buruli Ulcer ◽

Oxidase Inhibitor ◽

Mycobacterium Ulcerans ◽

Infection Model ◽

Synergistic Activity ◽

Content Type ◽

Drug Regimens ◽

Long Acting ◽

Synthase Inhibitor

ABSTRACT Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. However, STR requires injections and may be toxic, and CLR shares an adverse drug-drug interaction with RIF and may be poorly tolerated. Studies in a mouse footpad infection model showed that increasing the dose of RIF or using the long-acting rifamycin rifapentine (RPT), in combination with clofazimine (CFZ), a relatively well-tolerated antibiotic, can shorten treatment to 4 weeks. CFZ is reduced by a component of the electron transport chain (ETC) to produce reactive oxygen species toxic to bacteria. Synergistic activity of CFZ with other ETC-targeting drugs, the ATP synthase inhibitor bedaquiline (BDQ) and the bc1:aa3 oxidase inhibitor Q203 (now named telacebec), was recently described against Mycobacterium tuberculosis. Recognizing that M. tuberculosis mutants lacking the alternative bd oxidase are hypersusceptible to Q203 and that Mycobacterium ulcerans is a natural bd oxidase-deficient mutant, we tested the in vitro susceptibility of M. ulcerans to Q203 and evaluated the treatment-shortening potential of novel 3- and 4-drug regimens combining RPT, CFZ, Q203, and/or BDQ in a mouse footpad model. The MIC of Q203 was extremely low (0.000075 to 0.00015 μg/ml). Footpad swelling decreased more rapidly in mice treated with Q203-containing regimens than in mice treated with RIF and STR (RIF+STR) and RPT and CFZ (RPT+CFZ). Nearly all footpads were culture negative after only 2 weeks of treatment with regimens containing RPT, CFZ, and Q203. No relapse was detected after only 2 weeks of treatment in mice treated with any of the Q203-containing regimens. In contrast, 15% of mice receiving RIF+STR for 4 weeks relapsed. We conclude that it may be possible to cure patients with Buruli ulcer in 14 days or less using Q203-containing regimens rather than currently recommended 56-day regimens.

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In Vitro Susceptibility ofMycobacterium ulceransIsolates to Selected Antimicrobials

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2017/5180984 ◽

2017 ◽

Vol 2017 ◽

pp. 1-6

Author(s):

Enid Owusu ◽

Mercy J. Newman ◽

Kwesi K. Addo ◽

Phyllis Addo

Keyword(s):

Inhibitory Activity ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Drug Misuse ◽

Definitive Treatment ◽

Dilution Method ◽

Agar Dilution Method ◽

In Vitro Susceptibility ◽

Antimicrobial Drug Resistance

Background.The current definitive treatment of Buruli ulcer with antibiotics makes the issue of antimicrobial drug resistance an unavoidable one. This is as a result of drug misuse by health personnel and patients’ noncompliance to treatment regimen. Monitoring of these factors and screening for new effective antimicrobials are crucial to effective management of Buruli ulcer disease. This study therefore investigated the inhibitory activity of some antibiotics against isolates ofMycobacterium ulcerans.Methods.Activity of eight antibiotics was tested against twelveM. ulceransisolates (2 reference strains and 10 clinical isolates). The anti-M. ulceransactivities were determined by the agar dilution method and the minimum inhibitory concentrations (MICs) were determined by the agar proportion method.Results.All antimicrobials investigated had activity againstM. ulceransisolates tested. The MICs ranged from 0.16 μg/mL to 2.5 μg/mL. Azithromycin recorded the highest inhibitory activity at a mean MIC of 0.39 μg/mL, whilst clofazimine a second-line antileprosy drug, recorded the lowest at a mean MIC of 2.19 μg/mL. Among the four antituberculosis drugs, rifampicin had the highest activity with a mean MIC of 0.81 μg/mL.Conclusion.Azithromycin could be considered as a lucrative alternative to existing treatment methods for inhibitingM. ulceransin Ghana.

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Investigation of skin microbiota reveals Mycobacterium ulcerans-Aspergillus sp. trans-kingdom communication

10.1101/869636 ◽

2019 ◽

Cited By ~ 1

Author(s):

N. Hammoudi ◽

C Cassagne ◽

M. Million ◽

S Ranque ◽

O. Kabore ◽

...

Keyword(s):

Burkina Faso ◽

Aspergillus Flavus ◽

Dna Sequences ◽

Buruli Ulcer ◽

Opportunistic Pathogen ◽

Negative Control ◽

Mycobacterium Ulcerans ◽

Skin Microbiota ◽

Endemic Region

ABSTRACTBackgroundMycobacterium ulcerans secrete a series of non-ribosomal-encoded toxins known as mycolactones that are responsible for causing a disabling ulceration of the skin and subcutaneous tissues named Buruli ulcer. The disease is the sole non-contagion among the three most common mycobacterial diseases in humans. Direct contact with contaminated wetlands is a risk factor for Buruli ulcer, responsible for M. ulcerans skin carriage before transcutaneous inoculation with this opportunistic pathogen.Methodology and principal findingsIn this study, we analysed the bacterial and fungal skin microbiota in individuals exposed to M. ulcerans in Burkina Faso. We showed that M. ulcerans-specific DNA sequences were detected on the unbreached skin of 6/52 (11.5%) asymptomatic farmers living in Sindou versus 0/52 (0%) of those living in the non-endemic region of Tenkodogo. Then, we cultured the skin microbiota of asymptomatic M. ulcerans carriers and negative control individuals, all living in the region of Sindou. A total of 84 different bacterial and fungal species were isolated, 21 from M. ulcerans-negative skin samples, 31 from M. ulcerans-positive samples and 32 from both. More specifically, Actinobacteria, Aspergillus niger and Aspergillus flavus were significantly associated with M. ulcerans skin carriage. We further observed that in vitro, mycolactones induced spore germination of A. flavus, attracting the fungal network.ConclusionThese unprecedented observations suggest that interactions with fungi may modulate the outcome of M. ulcerans skin carriage, opening new venues to the understanding of Buruli ulcer pathology, prophylaxis and treatment of this still neglected tropical infection.Author summaryBuruli ulcer is a chronic infectious disease caused by the environmental opportunistic pathogen Mycobacterium ulcerans which secretes an exotoxin responsible for its pathogenicity. The reservoir and sources of M. ulcerans in the environment remain elusive and its mode of transmission is unclear. To acquire M. ulcerans infection, at least two conditions must be met, viable bacteria and a skin lesion as demonstrated by experimental animal models. In this study, we showed that M. ulcerans specific DNA sequences could be detected on the healthy skin of asymptomatic farmers living in one region of Burkina Faso where Buruli ulcer cases had already been reported, but not in Buruli ulcer-free regions, suggesting skin carriage after contacts with environmental sources. We also investigated the skin microbiota of M. ulcerans carriers and found significant associations of some bacteria and fungi with skin carriage of M. ulcerans. These associations may due to the effect of mycolactones on some fungi species. As we showed previously with Mucor circinelloides and here with Aspergillus flavus.

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Aberrant stromal tissue factor and mycolactone-driven vascular permeability, exacerbated by IL-1β, orchestrate pathogenic fibrin formation in Buruli ulcer lesions

10.1101/2021.08.04.21261598 ◽

2021 ◽

Author(s):

Louise Tzung-Harn Hsieh ◽

Scott J Dos Santos ◽

Joy Ogbechi ◽

Aloysius D Loglo ◽

Francisco J Salguero ◽

...

Keyword(s):

Tissue Factor ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

In Vitro Assays ◽

Platelet Glycoprotein ◽

Fibrin Deposition ◽

Clotting Factors ◽

Extrinsic Pathway ◽

Fibrin Formation

The neglected tropical disease Buruli ulcer, caused by Mycobacterium ulcerans infection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to the M. ulcerans exotoxin mycolactone depletes the expression of thrombomodulin and impacts anticoagulation at the endothelial cell surface. Moreover, while widespread fibrin deposition is a common feature of BU lesions, the cause of this phenotype is not clear. Here, we performed sequential staining of serial tissue sections of BU patient biopsies and unbiased analysis of up to 908 individual non-necrotic vessels of eight BU lesions to investigate its origins. Most vessels showed evidence of endothelial dysfunction being thrombomodulin-negative, von Willebrand factor-negative and/or had endothelium that stained positively for tissue factor (TF). Primary haemostasis was rarely evident by platelet glycoprotein CD61 staining. Localisation of TF in these lesions was complex and aberrant, including diffuse staining of the stroma some distance from the basement membrane and TF-positive infiltrating cells (likely eosinophils). This pattern of abnormal TF staining was the only phenotype that was significantly associated with fibrin deposition, and its extent correlated significantly with the distance that fibrin deposition extended into the tissue. Hence, fibrin deposition in Buruli ulcer lesions is likely driven by the extrinsic pathway of coagulation. To understand how this could occur, we investigated whether clotting factors necessary for fibrin formation might gain access to the extravascular compartment due to loss of the vascular barrier. In vitro assays using primary vascular and lymphatic endothelial cells showed that mycolactone increased the permeability of monolayers to dextran within 24 hours. Moreover, co-incubation of cells with interleukin-1β exacerbated mycolactones effects, nearly doubling the permeability of the monolayer compared to each challenge alone. We propose that leaky vascular and lymphatic systems are important drivers of extravascular fibrin deposition, necrosis and oedema frequently seen in Buruli ulcer patients.

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In Vitro and In Vivo Activities of Rifampin, Streptomycin, Amikacin, Moxifloxacin, R207910, Linezolid, and PA-824 against Mycobacterium ulcerans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00052-06 ◽

2006 ◽

Vol 50 (6) ◽

pp. 1921-1926 ◽

Cited By ~ 83

Author(s):

Baohong Ji ◽

Sébastien Lefrançois ◽

Jerome Robert ◽

Aurélie Chauffour ◽

Chantal Truffot ◽

...

Keyword(s):

Clinical Trial ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Potential Toxicity ◽

Peak Serum Level ◽

Bactericidal Effects ◽

The Mean ◽

The Cost

ABSTRACT Seven antimicrobials were tested in vitro against 29 clinical isolates of Mycobacterium ulcerans. R207910 demonstrated the lowest MIC50 and MIC90, followed by moxifloxacin (MXF), streptomycin (STR), rifampin (RIF), amikacin (AMK), linezolid (LZD), and PA-824. All but PA-824 demonstrated an MIC90 significantly less than the clinically achievable peak serum level. Administered as monotherapy to mice, RIF, STR, AMK, MXF, R207910, and LZD demonstrated some degree of bactericidal activity, whereas PA-824 failed to prevent mortality and to reduce the mean number of CFU in the footpads. Because 4 or 8 weeks of treatment by the combinations RIF-MXF, RIF-R207910, and RIF-LZD displayed bactericidal effects similar to those of RIF-STR and RIF-AMK, these three combinations might be considered as orally administered combined regimens for treatment of Buruli ulcer. Taking into account the cost, potential toxicity, and availability, the combination RIF-MXF appears more feasible for application in the field; additional experiments with mice are warranted to define further its activity against M. ulcerans. In addition, a pilot clinical trial is proposed to test the efficacy of RIF-MXF for treatment of Buruli ulcer.

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