Detection of Mycobacterium ulcerans DNA in the Environment,Ivory Coast

Background

Ivory Coast is a West African country with the highest reported cases of Buruli ulcer, a disabling subcutaneous infection due to Mycobacterium ulcerans. However, the prevalence of environmental M. ulcerans is poorly known in this country.

Methods

We collected 496 environmental specimens consisting of soil (n = 100), stagnant water (n = 200), plants (n = 100) and animal feces (n = 96) in Ivory Coast over five months in the dry and wet seasons in regions which are free of Buruli ulcer (control group A; 250 specimens) and in regions where the Buruli ulcer is endemic (group B; 246 specimens). After appropriate total DNA extraction incorporating an internal control, the M. ulcerans IS2404 and KR-B gene were amplified by real-time PCR in samples. In parallel, a calibration curve was done for M. ulcerans Agy99 IS2404 and KR-B gene.

Results

Of 460 samples free of PCR inhibition, a positive real-time PCR detection of insertion sequence IS2404 and KR-B gene was observed in 1/230 specimens in control group A versus 9/230 specimens in group B (P = 0.02; Fisher exact test). Positive specimens comprised seven stagnant water specimens, two feces specimens confirmed to be of Thryonomys swinderianus (agouti) origin by real-time PCR of the cytb gene; and one soil specimen. Extrapolation from the calibration curves indicated low inoculums ranging from 1 to 10² mycobacteria/mL.

Conclusion

This study confirms the presence of M. ulcerans in the watery environment surrounding patients with Buruli ulcer in Ivory Coast. It suggests that the agouti, which is in close contacts with populations, could play a role in the environmental cycle of M. ulcerans, as previously suggested for the closely related possums in Australia.     

Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa.

Methodology/Principal Findings

We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities.

Conclusions/Significance

This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.     

Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer

Background

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters.

Methodology/Principal Findings

To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a T_H1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model.

Conclusions

Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.     

Detection of Mycobacterium ulcerans by the loop mediated isothermal amplification method

Background

Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent.

Methodology

We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR.

Principal Findings

The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used.

Conclusion/Significance

The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU     

Development of a Dry-Reagent-Based qPCR to Facilitate the Diagnosis of Mycobacterium ulcerans Infection in Endemic Countries

Background

Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.

Methodology/Principal Findings

We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix.

Conclusions

Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

Background

As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.

Methodology/Principal Findings

Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.

Conclusions/Significance

Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.     

Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.     

Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs

Background

Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.

Methodology/Principal Findings

Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.

Conclusion

In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.     

Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity

Background

Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes—key cells in wound healing—and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS.

Methodology/Principal Findings

The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe²⁺, completely prevented mycolactone mediated cytotoxicity.

Conclusions/Significance

This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.     

First cultivation and characterization of Mycobacterium ulcerans from the environment

Background

Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.

Methodology/Principal Findings

One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5′ end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3′ end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.

Conclusion

Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.     

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer

Background

Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes.

Methodology

We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin.

Principal Findings

The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse.

Conclusions

To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.     

Secondary Buruli Ulcer Skin Lesions Emerging Several Months after Completion of Chemotherapy: Paradoxical Reaction or Evidence for Immune Protection?

Background

The neglected tropical disease Buruli ulcer (BU) caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic ulcerative skin lesions. Histopathological features are progressive tissue necrosis, extracellular clusters of acid fast bacilli (AFB) and poor inflammatory responses at the site of infection. After the recommended eight weeks standard treatment with rifampicin and streptomycin, a reversal of the local immunosuppression caused by the macrolide toxin mycolactone of M. ulcerans is observed.

Methodology/Principal Findings

We have conducted a detailed histopathological and immunohistochemical analysis of tissue specimens from two patients developing multiple new skin lesions 12 to 409 days after completion of antibiotic treatment. Lesions exhibited characteristic histopathological hallmarks of Buruli ulcer and AFB with degenerated appearance were found in several of them. However, other than in active disease, lesions contained massive leukocyte infiltrates including large B-cell clusters, as typically found in cured lesions.

Conclusion/Significance

Our histopathological findings demonstrate that the skin lesions emerging several months after completion of antibiotic treatment were associated with M. ulcerans infection. During antibiotic therapy of Buruli ulcer development of new skin lesions may be caused by immune response-mediated paradoxical reactions. These seem to be triggered by mycobacterial antigens and immunostimulators released from clinically unrecognized bacterial foci. However, in particular the lesions that appeared more than one year after completion of antibiotic treatment may have been associated with new infection foci resolved by immune responses primed by the successful treatment of the initial lesion.     

Photodegradation of the Mycobacterium ulcerans toxin, mycolactones: considerations for handling and storage

Background

Mycolactones are toxins secreted by M. ulcerans, the etiological agent of Buruli ulcer. These toxins, which are the main virulence factors of the bacilli, are responsible for skin lesions. Considering their specificity for M. ulcerans and their presence in skin lesions even at early stages, mycolactones are promising candidates for the development of a diagnostic tool for M. ulcerans infection. Stability of purified mycolactones towards light and heat has not yet been investigated, despite the importance of such parameters in the selection of strategies for a diagnosis tool development. In this context, the effects of UV, light and temperature on mycolactone stability and biological activity were studied.

Methodology/Principal Findings

To investigate the effect of these physical parameters, mycolactones were exposed to different wavelengths in several solvents and temperatures. Structural changes and biological activity were monitored. Whilst high temperature had no effect on mycolactones, UV irradiation (UV-A, UV-B and UV-C) and sunlight exposure caused a considerable degradation, as revealed by LC-MS and NMR analysis, correlated with a loss of biological activity. Moreover, effect of UVs on mycolactone caused a photodegradation rather than a phototransformation due to the identification of degradation product.

Conclusion/Significance

This study demonstrates the high sensitivity of mycolactones to UVs as such it defines instructions for storage and handling.     

Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

Background

Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone.

Methodology/Principal Findings

A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na⁺] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease.

Conclusions/Significance

Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration.     

Structure-Activity Relationship Studies on the Macrolide Exotoxin Mycolactone of Mycobacterium ulcerans

Background

Mycolactones are a family of polyketide-derived macrolide exotoxins produced by Mycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. The toxin is synthesized by polyketide synthases encoded by the virulence plasmid pMUM. The apoptotic, necrotic and immunosuppressive properties of mycolactones play a central role in the pathogenesis of M. ulcerans.

Methodology/Principal Findings

We have synthesized and tested a series of mycolactone derivatives to conduct structure-activity relationship studies. Flow cytometry, fluorescence microscopy and Alamar Blue-based metabolic assays were used to assess activities of mycolactones on the murine L929 fibroblast cell line. Modifications of the C-linked upper side chain (comprising C12–C20) caused less pronounced changes in cytotoxicity than modifications in the lower C5-O-linked polyunsaturated acyl side chain. A derivative with a truncated lower side chain was unique in having strong inhibitory effects on fibroblast metabolism and cell proliferation at non-cytotoxic concentrations. We also tested whether mycolactones have antimicrobial activity and found no activity against representatives of Gram-positive (Streptococcus pneumoniae) or Gram-negative bacteria (Neisseria meningitis and Escherichia coli), the fungus Saccharomyces cerevisae or the amoeba Dictyostelium discoideum.

Conclusion

Highly defined synthetic compounds allowed to unambiguously compare biological activities of mycolactones expressed by different M. ulcerans lineages and may help identifying target structures and triggering pathways.     

Seasonal and Regional Dynamics of M. ulcerans Transmission in Environmental Context: Deciphering the Role of Water Bugs as Hosts and Vectors

Background

Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies.

Methodology/Principal Findings

The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas.

Conclusion/Significance

The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.     

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/Principal Finding

Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/Significance

Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.