scholarly journals Mycolactone-independent pathogenicity of Mycobacterium ulcerans: an experimental study in plants

2019 ◽  
Author(s):  
A. Bouam ◽  
M. Drancourt
Keyword(s):  
Buruli Ulcer ◽  
Negative Control ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Tomato Plants ◽  
Cellular Effects ◽  
Infected Root ◽  
Significant Difference ◽  
Negative Controls ◽  
Intact Roots

ABSTRACTMycobacterium ulcerans, the etiologic agent of Buruli ulcer in humans and animals, secretes macrolide exotoxins mycolactones which damage tissues after a cascade of cellular effects. M. ulcerans, an environmental organism with still elusive reservoirs and sources has been detected in soil and water in endemic areas where it could be in contact with plants. Symptom observations, microscopy and molecular biology were used to investigate M. ulcerans contact with plants in an experimental model mimicking the known pathology of Buruli ulcer in humans. Solanum lycopereum (tomato) plants with scarified or intact roots were transplanted into pots containing contaminated soil with M. ulcerans or a mixture of mycolactones A/B and C in the presence of negative control groups. Whereas plants with intact roots remained asymptomatic, M. ulcerans-infected plants with scarified roots had significantly more diseased leaves than controls (p = 0.004). Optic microscopy examination showed significantly more mycobacteria in the secondary and main roots than in controls (p=0.0008). Real-time PCRs detected M. ulcerans DNA in 7/12 (58%) of infected root samples versus none in the control plants (p = 0.04). Further study of plants with mycolactones A/B and C yielded no significant difference with negative controls. These results suggest that in this model, M. ulcerans exhibits a mycolactone-independent pathogenicity whose mechanism remains to be elucidated.

scholarly journals Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

2009 ◽  
Vol 75 (17) ◽  
pp. 5667-5675 ◽  
Author(s):  
Michael K�ser ◽  
Julia Hauser ◽  
Pamela Small ◽  
Gerd Pluschke
Keyword(s):  
Buruli Ulcer ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Nucleotide Polymorphisms ◽  
Differentiation Markers ◽  
Single Nucleotide ◽  
Virulence Plasmids ◽  
Sequence Polymorphisms ◽  
Genetic Features ◽  
Relationship Of

ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

scholarly journals Snapshot fecal survey of domestic animals in rural Ghana forMycobacterium ulcerans

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2065 ◽  
Author(s):  
Nicholas J. Tobias ◽  
Nana Ama Ammisah ◽  
Evans K. Ahortor ◽  
John R. Wallace ◽  
Anthony Ablordey ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Buruli Ulcer ◽  
Domestic Animals ◽  
Mycobacterium Ulcerans ◽  
Animal Reservoir ◽  
Rural Ghana ◽  
Reservoir Species ◽  
Mode Of Transmission ◽  
High Concentrations ◽  
Negative Controls

Identifying the source reservoirs ofMycobacterium ulceransis key to understanding the mode of transmission of this pathogen and controlling the spread of Buruli ulcer (BU). In Australia, the native possum can harborM. ulceransin its gastrointestinal tract and shed high concentrations of the bacteria in its feces. To date, an analogous animal reservoir in Africa has not been identified. Here we tested the hypothesis that common domestic animals in BU endemic villages of Ghana are reservoir species analogous to the Australian possum. Using linear-transects at 10-meter intervals, we performed systematic fecal surveys across four BU endemic villages and one non-endemic village in the Asante Akim North District of Ghana. One hundred and eighty fecal specimens from a single survey event were collected and analyzed by qPCR for theM. ulceransdiagnostic DNA targets IS2404and KR-B. Positive and negative controls performed as expected but all 180 test samples were negative. This structured snapshot survey suggests that common domestic animals living in and around humans do not shedM. ulceransin their feces. We conclude that, unlike the Australian native possum, domestic animals in rural Ghana are unlikely to be major reservoirs ofM. ulcerans.

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

2019 ◽  
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.

scholarly journals Use of the Immunodominant 18-Kilodalton Small Heat Shock Protein as a Serological Marker for Exposure to Mycobacterium ulcerans

2006 ◽  
Vol 13 (12) ◽  
pp. 1314-1321 ◽  
Author(s):  
Diana Diaz ◽  
Heinz Döbeli ◽  
Dorothy Yeboah-Manu ◽  
Ernestina Mensah-Quainoo ◽  
Arno Friedlein ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Serological Test ◽  
Buruli Ulcer ◽  
Small Heat Shock Protein ◽  
Cross Reactivity ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Target Antigen ◽  
Mycobacterial Species

ABSTRACT While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

scholarly journals Evidence for an Intramacrophage Growth Phase of Mycobacterium ulcerans

2006 ◽  
Vol 75 (2) ◽  
pp. 977-987 ◽  
Author(s):  
Egídio Torrado ◽  
Alexandra G. Fraga ◽  
António G. Castro ◽  
Pieter Stragier ◽  
Wayne M. Meyers ◽  
...  
Keyword(s):  
Buruli Ulcer ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Host Cells ◽  
Delayed Type Hypersensitivity ◽  
In Vitro Assays ◽  
Mouse Macrophages ◽  
Inflammatory Infiltrates ◽  
Extracellular Location

ABSTRACT Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
N. Hammoudi ◽  
C. Cassagne ◽  
M. Million ◽  
S. Ranque ◽  
O. Kabore ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Buruli Ulcer ◽  
Opportunistic Pathogen ◽  
Fungal Species ◽  
Negative Control ◽  
Mycobacterium Ulcerans ◽  
Skin Microbiota ◽  
Endemic Region ◽  
Aspergillus Sp

AbstractMycobacterium 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. In 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. These 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.

scholarly journals High antibody titres induced by protein subunit vaccines using Mycobacterium ulcerans antigens Hsp18 and MUL_3720 with a TLR-2 agonist fail to protect against Buruli ulcer in mice

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9659
Author(s):  
Kirstie M. Mangas ◽  
Nicholas J. Tobias ◽  
Estelle Marion ◽  
Jérémie Babonneau ◽  
Laurent Marsollier ◽  
...  
Keyword(s):  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Infection Model ◽  
Protein Subunit ◽  
Subunit Vaccines ◽  
Significant Difference ◽  
Vaccine Antigens ◽  
Antibody Titres ◽  
Vaccine Potential ◽  
Time To Onset

Background Mycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. Methods Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. Results The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. Conclusions These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

Thrombogenicity Testing of Medical Devices in a Minimally Heparinized Ovine Blood Loop

2017 ◽  
Vol 11 (2) ◽  
Author(s):  
Kent Grove ◽  
Steve M. Deline ◽  
Tim F. Schatz ◽  
Sarah E. Howard ◽  
Deanna Porter ◽  
...  
Keyword(s):  
Medical Devices ◽  
Selection Process ◽  
Negative Control ◽  
In Vitro Assay ◽  
In Vivo Models ◽  
Vitro Assay ◽  
Significant Difference ◽  
Negative Controls

ISO 10993-4 in vivo thrombogenicity testing is frequently performed for regulatory approval of many blood-contacting medical devices and is often a key part of submission packages. Given the current state of in vivo thrombogenicity assays, a more robust and reproducible assay design, including in vitro models, is needed. This study describes an in vitro assay that integrates freshly harvested ovine blood containing minimal heparin in a closed pumped loop. To confirm the reproducibility of this assay, control materials were identified that elicited either a positive or a negative thrombogenic response. These controls demonstrated reproducibility in the resulting thrombogenicity scores with median scores of 5 and 0 for the positive and negative controls, respectively, which also demonstrated a significant difference (p < 0.0001). For a direct comparison of the in vitro blood loop assay to the traditional in vivo nonanticoagulated venous implant (NAVI) assay, seven sheep were used as blood donors for the loop and then as subjects for an NAVI assay. In each assay—loop or NAVI—three study articles were used: the positive and negative controls and a marketed, approved catheter. The resulting thrombogenicity scores were similar when comparing the loop to the NAVI results. For each study article, the median thrombogenicity scores were the same in these two different assays, being 0, 1, and 5 for the negative control, the marketed catheter, and the positive control, respectively. These data suggest that the in vitro assay performs similarly to the in vivo NAVI assay. This in vitro blood loop method has the potential to predict a materials' in vivo thrombogenicity, can substantially de-risk the materials or coating selection process, and may eventually be able to replace the in vivo models currently in use.

scholarly journals Evaluation of Apical Microleakage in Open Apex Teeth Using MTA Apical Plug in Different Sessions

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Mohammad Yazdizadeh ◽  
Zeinab Bouzarjomehri ◽  
Navid Khalighinejad ◽  
Leyli Sadri
Keyword(s):  
Methylene Blue ◽  
Nitric Acid ◽  
Methyl Salicylate ◽  
Negative Control Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Dye Penetration ◽  
Significant Difference ◽  
Negative Controls

Aim. To compare microleakage of apexification using MTA in one or two sessions. Materials and Methods. 88 single rooted teeth were prepared and divided into two groups then received MTA apical plug. In the first group, the teeth were immersed in normal saline for 24 hours and then backfilled with guttapercha and AH26 sealer. In the second group, the teeth were obturated immediately after receiving apical plug. Four positive and four negative controls were selected. All specimens were placed in 1% methylene blue and decalcified in 5% nitric acid and finally were placed in methyl salicylate until getting transparent. All teeth were visualized for assessment of dye penetration under stereo dissecting microscope. Results. 36 and 35 teeth showed dye leakage in the first and second groups. Dye penetration into the entire canal length was confirmed in the positive control group, and in the negative control group no dye penetration was seen. Mean dye penetration in the first and second group was 5813 and 9152 μm. -test revealed a significant difference between dye penetrations of two groups (). Conclusion. MTA requires adequate time for setting in the presence of the moisture, and final obturation should be delayed until final setting of MTA.

scholarly journals High antibody titres induced by protein subunit vaccines against Buruli ulcer using Mycobacterium ulcerans antigens Hsp18 and MUL_3720

2020 ◽  
Author(s):  
Kirstie M. Mangas ◽  
Nicholas Tobias ◽  
Estelle Marion ◽  
Jérémie Babonneau ◽  
Laurent Marsollier ◽  
...  
Keyword(s):  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Infection Model ◽  
Protein Subunit ◽  
Subunit Vaccines ◽  
Significant Difference ◽  
Vaccine Antigens ◽  
Antibody Titres ◽  
Vaccine Potential ◽  
Time To Onset

AbstractBackgroundMycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU.MethodsRecombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice.ResultsThe MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals.ConclusionsThese data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

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