@article {13, title = {A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens.}, journal = {J Microbiol Methods}, volume = {76}, year = {2009}, month = {2009 Feb}, pages = {152-8}, abstract = {

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens.

}, keywords = {Animals, Buruli Ulcer, DNA Transposable Elements, DNA, Bacterial, DNA, Plant, Environmental Monitoring, Humans, Mycobacterium ulcerans, Polymerase Chain Reaction, Reagent Kits, Diagnostic, Sensitivity and Specificity, Soil Microbiology, Water Microbiology}, issn = {0167-7012}, doi = {10.1016/j.mimet.2008.10.002}, author = {Durnez, Lies and Stragier, Pieter and Roebben, Karen and Ablordey, Anthony and Leirs, Herwig and Portaels, Fran{\c c}oise} } @article {2, title = {Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units.}, journal = {J Bacteriol}, volume = {187}, year = {2005}, month = {2005 Mar}, pages = {1639-47}, abstract = {

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.

}, keywords = {Bacterial Typing Techniques, Base Sequence, Genotype, Interspersed Repetitive Sequences, Minisatellite Repeats, Molecular Sequence Data, Mycobacterium marinum, Mycobacterium ulcerans, Phylogeny, Polymorphism, Genetic, Sequence Alignment, Sequence Homology, Nucleic Acid, Species Specificity}, issn = {0021-9193}, doi = {10.1128/JB.187.5.1639-1647.2005}, author = {Stragier, Pieter and Ablordey, Anthony and Meyers, Wayne M and Portaels, Fran{\c c}oise} } @article {3, title = {Comparative nucleotide sequence analysis of polymorphic variable-number tandem-repeat Loci in Mycobacterium ulcerans.}, journal = {J Clin Microbiol}, volume = {43}, year = {2005}, month = {2005 Oct}, pages = {5281-4}, abstract = {

We analyzed a set of variable-number tandem-repeat (VNTR) loci to assess their nucleotide sequence diversity in isolates of three Mycobacterium ulcerans genotypes. Sequence variants in two loci resulted in intraspecies resolution of Southeast Asian and Asian genotypes in contrast to a homogenous sequence composition among African isolates. Nucleotide sequence polymorphism in repeat units can enhance discrimination of VNTR loci.

}, keywords = {Bacterial Typing Techniques, Base Sequence, Genetic Variation, Genotype, Humans, Minisatellite Repeats, Molecular Sequence Data, Mycobacterium ulcerans, Polymorphism, Genetic, Sequence Alignment, Species Specificity}, issn = {0095-1137}, doi = {10.1128/JCM.43.10.5281-5284.2005}, author = {Ablordey, Anthony and Hilty, Markus and Stragier, Pieter and Swings, Jean and Portaels, Fran{\c c}oise} } @article {5, title = {Heterogeneity among Mycobacterium ulcerans isolates from Africa.}, journal = {Emerg Infect Dis}, volume = {12}, year = {2006}, month = {2006 May}, pages = {844-7}, abstract = {

Mycobacterium ulcerans causes Buruli ulcer, an ulcerative skin disease in tropical and subtropical areas. Despite restricted genetic diversity, mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis on M. ulcerans revealed 3 genotypes from different African countries. It is the first time this typing method succeeded directly on patient samples.

}, keywords = {Africa, Bacterial Typing Techniques, Base Sequence, Genetic Variation, Genotype, Humans, Molecular Epidemiology, Molecular Sequence Data, Mycobacterium Infections, Nontuberculous, Mycobacterium ulcerans, Phylogeny, Polymorphism, Genetic, Tandem Repeat Sequences}, issn = {1080-6040}, doi = {10.3201/eid1205.051191}, author = {Stragier, Pieter and Ablordey, Anthony and Bayonne, L Manou and Lugor, Yatta L and Sindani, Ireneaus S and Suykerbuyk, Patrick and Wabinga, Henry and Meyers, Wayne M and Portaels, Fran{\c c}oise} } @article {7, title = {Mycobacterium liflandii infection in European colony of Silurana tropicalis.}, journal = {Emerg Infect Dis}, volume = {13}, year = {2007}, month = {2007 May}, pages = {743-6}, abstract = {

Mycobacterium liflandii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the first epizootic of mycobacteriosis in a European colony of clawed frogs (Silurana tropicalis), previously imported from a United States biologic supply company. Our findings suggest the emerging potential of this infection through international trade.

}, keywords = {Animals, Europe, Models, Animal, Mycobacterium, Mycobacterium Infections, United States, Xenopus, Zoonoses}, issn = {1080-6040}, doi = {10.3201/eid1305.060625}, author = {Suykerbuyk, Patrick and Vleminckx, Kris and Pasmans, Frank and Stragier, Pieter and Ablordey, Anthony and Tran, Hong Thi and Hermans, Katleen and Fleetwood, Michelle and Meyers, Wayne M and Portaels, Fran{\c c}oise} } @article {9, title = {VNTR analysis differentiates Mycobacterium ulcerans and IS2404 positive mycobacteria.}, journal = {Syst Appl Microbiol}, volume = {30}, year = {2007}, month = {2007 Nov}, pages = {525-30}, abstract = {

In recent years, numerous IS2404 positive mycobacteria have been identified, compromising the detection of Mycobacterium ulcerans. In this study, variable number of tandem repeats (VNTR) analysis was successfully applied on cultures and tissue specimens to differentiate all currently known IS2404 positive mycobacteria from M. ulcerans.

}, keywords = {Bacterial Typing Techniques, Chromosomes, Bacterial, Cluster Analysis, DNA Transposable Elements, Minisatellite Repeats, Mycobacterium}, issn = {0723-2020}, doi = {10.1016/j.syapm.2007.06.001}, author = {Stragier, Pieter and Ablordey, Anthony and Durnez, Lies and Portaels, Fran{\c c}oise} } @article {10, title = {First cultivation and characterization of Mycobacterium ulcerans from the environment.}, journal = {PLoS Negl Trop Dis}, volume = {2}, year = {2008}, month = {2008}, pages = {e178}, abstract = {

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 L{\"o}wenstein-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{\textquoteright} end of 16S rRNA gene of 00-1441 was 100\% identical to M. ulcerans and M. marinum, and the sequence of the 3{\textquoteright} 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.

}, keywords = {Animals, Bacterial Toxins, Cells, Cultured, Environmental Microbiology, Female, Foot, Genotype, Hemiptera, Macrolides, Macrophages, Mass Spectrometry, Mice, Mice, Inbred BALB C, Mycobacterium ulcerans, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S}, issn = {1935-2735}, doi = {10.1371/journal.pntd.0000178}, author = {Portaels, Fran{\c c}oise and Meyers, Wayne M and Ablordey, Anthony and Castro, Ant{\'o}nio G and Chemlal, Karim and de Rijk, Pim and Elsen, Pierre and Fissette, Krista and Fraga, Alexandra G and Lee, Richard and Mahrous, Engy and Small, Pamela L C and Stragier, Pieter and Torrado, Eg{\'\i}dio and Van Aerde, Anita and Silva, Manuel T and Pedrosa, Jorge} }