Miriam Eddyani

Potential Role for Fish in Transmission of Mycobacterium ulcerans Disease (Buruli Ulcer): an Environmental Study

ABSTRACT This study reports a potential role that fish may play in the transmission of Mycobacterium ulcerans disease (Buruli ulcer). Fish found positive for M. ulcerans DNA all appear to feed on insects or plankton and are believed to concentrate M. ulcerans from this usual food source. These observations provide additional data supporting our previous hypothesis on sources of M. ulcerans and modes of transmission.

On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

BackgroundMycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum, M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations (M. pseudoshottsii, M. shinshuense, M. liflandii and M. marinum), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum.ResultsA high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer.ConclusionsM. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.

Fine-Needle Aspiration, an Efficient Sampling Technique for Bacteriological Diagnosis of Nonulcerative Buruli Ulcer

ABSTRACT Invasive punch or incisional skin biopsy specimens are currently employed for the bacteriological confirmation of the clinical diagnosis of Buruli ulcer (BU), a cutaneous infectious disease caused by Mycobacterium ulcerans. The efficacy of fine-needle aspirates (FNA) using fine-gauge needles (23G by 25 mm) for the laboratory confirmation of BU was compared with that of skin tissue fragments obtained in parallel by excision or punch biopsy. In three BU treatment centers in Benin, both types of diagnostic material were obtained from 33 clinically suspected cases of BU and subjected to the same laboratory analyses: i.e., direct smear examination, IS2404 PCR, and in vitro culture. Twenty-three patients, demonstrating 17 ulcerative and 6 nonulcerative lesions, were positive by at least two tests and were therefore confirmed to have active BU. A total of 68 aspirates and 68 parallel tissue specimens were available from these confirmed patients. When comparing the sensitivities of the three confirmation tests between FNA and tissue specimens, the latter yielded more positive results, but only for PCR was this significant. When only nonulcerative BU lesions were considered, however, the sensitivities of the confirmation tests using FNA and tissue specimens were not significantly different. Our results show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in nonulcerative lesions.

Application of real-time PCR in Ghana, a Buruli ulcer-endemic country, confirms the presence of Mycobacterium ulcerans in the environment

This study reports the first successful application of real-time PCR for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), in Ghana, a BU-endemic country. Environmental samples and organs of small mammals were analyzed. The real-time PCR assays confirmed the presence of M. ulcerans in a water sample collected in a BU-endemic village in the Ashanti Region.

Primary Culture of Mycobacterium ulcerans from Human Tissue Specimens after Storage in Semisolid Transport Medium

ABSTRACT Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of Mycobacterium ulcerans was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for M. ulcerans DNA by an IS2404-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of M. ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field.

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening

ABSTRACT With the rising number of patients with human immunodeficiency virus (HIV)/AIDS in developing countries, the control of mycobacteria is of growing importance. Previous studies have shown that rodents and insectivores are carriers of mycobacteria. However, it is not clear how widespread mycobacteria are in these animals and what their role is in spreading them. Therefore, the prevalence of mycobacteria in rodents and insectivores was studied in and around Morogoro, Tanzania. Live rodents were trapped, with three types of live traps, in three habitats. Pieces of organs were pooled per habitat, species, and organ type (stratified pooling); these sample pools were examined for the presence of mycobacteria by PCR, microscopy, and culture methods. The mycobacterial isolates were identified using phenotypic techniques and sequencing. In total, 708 small mammals were collected, 31 of which were shrews. By pool prevalence estimation, 2.65% of the animals were carriers of mycobacteria, with a higher prevalence in the urban areas and in Cricetomys gambianus and the insectivore Crocidura hirta. Nontuberculous mycobacteria (Mycobacterium chimaera, M. intracellulare, M. arupense, M. parascrofulaceum, and Mycobacterium spp.) were isolated from C. gambianus, Mastomys natalensis, and C. hirta. This study is the first to report findings of mycobacteria in African rodents and insectivores and the first in mycobacterial ecology to estimate the prevalence of mycobacteria after stratified pool screening. The fact that small mammals in urban areas carry more mycobacteria than those in the fields and that potentially pathogenic mycobacteria were isolated identifies a risk for other animals and humans, especially HIV/AIDS patients, that have a weakened immune system.

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.

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.

Occurrence of Free-Living Amoebae in Communities of Low and High Endemicity for Buruli Ulcer in Southern Benin

ABSTRACT Buruli ulcer or Mycobacterium ulcerans disease occurs mainly in areas in proximity to standing or slowly running freshwater, habitats in which free-living amoebae occur. For this reason, a possible link between the habitat of M. ulcerans and free-living amoebae was investigated. Free-living amoebae and mycobacteria were isolated from water and biofilm specimens taken from protected and unprotected sources of water in villages known to have either high or low endemicity for Buruli ulcer in Benin. Amoebae were isolated from 78.8% of samples. A greater proportion of water bodies in areas of high endemicity had amoebae than in areas of low endemicity (83.3% versus 66.7%). Protected sources of water were significantly more likely to contain amoebae in areas of high endemicity than in areas of low endemicity (88.0% versus 11.1%). Several pathogenic free-living amoebae and mycobacteria were isolated. However, no M. ulcerans was isolated and no specimen was positive for IS2404 PCR. Our results show that the study area has a water hygiene problem, which is greater in areas of high Buruli ulcer endemicity than in areas of low endemicity. Our observations indicate that additional studies are required to explore the possible link between free-living amoebae and mycobacteria.

Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

ABSTRACT Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.