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1 Swiss Tropical Institute, 4002 Basel, Switzerland
2 Department of Microbiology, Monash University, Clayton, Victoria, Australia
Correspondence
Markus Hilty
m.hilty{at}imperial.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Imperial College, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), causes Buruli ulcer, which is, after tuberculosis and leprosy, the third most important mycobacterial disease worldwide (Sizaire et al., 2006; van der Werf et al., 2003). Buruli ulcer is mainly prevalent in West Africa but cases have also been reported from various other tropical and sub-tropical areas of the world (Asiedu et al., 2000; Johnson et al., 2005). Genetic analysis indicates the emergence of M. ulcerans from M. marinum, a fish pathogen which can also cause disease in humans (Boddinghaus et al., 1990; Tonjum et al., 1998). There is a strong association between the occurrence of M. ulcerans infection in humans and slow-flowing or stagnant water bodies (Marsollier et al., 2007; Portaels et al., 1999; Roberts & Hirst, 1997; Ross et al., 1997; Stinear et al., 2000a). Aquatic snails (Marsollier et al., 2004), fish (Eddyani et al., 2004) and water bugs (Marsollier et al., 2002) are currently being discussed as passive and active hosts, respectively. However, the exact mode of transmission of Buruli ulcer has remained a mystery, partly because no genetic fingerprinting tool is available for micro-epidemiological analysis of infection chains.
Variable number tandem repeats (VNTR) typing is a promising typing tool for M. ulcerans, because analysis based on few loci already has a higher discriminatory power than other standard molecular typing methods (Ablordey et al., 2005a, b; Hilty et al., 2006; Stragier et al., 2005, 2006). VNTR typing thus revealed for the first time genetic diversity within isolates from African countries (Hilty et al., 2006; Stragier et al., 2006). So far, two approaches have been used to identify loci suitable for VNTR typing of M. ulcerans. These are the screening of the Mycobacterium marinum genome sequence for tandem repeats containing loci with tandem repeats finder software (Ablordey et al., 2005b; Hilty et al., 2006), and BLAST searches for homologues of M. tuberculosis mycobacterial interspersed repetitive units in M. ulcerans (Stragier et al., 2005). Here, we have searched for the first time the completely assembled M. ulcerans genome sequence for tandem repeats containing loci and have evaluated the potential of the identified loci for VNTR-based typing.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Primer design.
Specific primers targeting repeat-flanking genomic sequences of M. ulcerans Agy99 were designed using Frodo Primer 3 software (http://frodo.wi.mit.edu/). According to sequence information from the M. marinum genome database (www.sanger.ac.uk/projects/M_marinum), the M. marinum sequences corresponding to these primers were 100 % identical, except for four primers, 1676, 1682, 1684 and 1692, which contained single nucleotide mismatches (Table 1).
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) was performed as described by Hilty et al. (2006). Briefly, PCR mixtures (final volume, 20 µl) contained 2 µl Taq PCR buffer, deoxynucleoside triphosphates (0.2 mM each), 1 U AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems), each primer at a concentration of 0.5 µM and mycobacterial DNA. PCR was performed for 40 cycles of 0.5 min at 94 °C, 0.5 min at 65 °C and 1 min at 72 °C. The reactions were terminated by incubation for 10 min at 72 °C. PCR fragments were analysed by agarose gel electrophoresis using 2 % NuSieve agarose, and sizes of the amplicons were estimated by comparison with Size Marker VIII (Roche), compared with the product size of Agy99 and copy number designated.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, b; Hilty et al., 2006; Stragier et al., 2005, 2006). Eleven out of 45 loci were excluded from further analysis for various reasons, including the presence of insertion sequence elements. For the remaining 34 loci, primers were designed (Table 1) and PCR amplification of a panel of 11 well-characterized M. ulcerans clinical isolates of diverse geographical origins performed. As it was our primary objective to develop a genetic fingerprinting method for African M. ulcerans isolates, more strains from Africa (6) than from other continents were analysed.
All 34 loci showed polymorphisms within the 11 isolates tested, but only alleles with one and with two repeats were found. Based on the typing results, the new VNTR loci were classified into four different subgroups, designated i–iv (Table 2). While subgroups i and ii comprised a multitude of loci (17 and 15, respectively), subgroups iii and iv were each represented by only one locus. For all 34 new VNTR loci, the M. marinum genome sequencing strain and the M. ulcerans isolates from Japan and French Guyana had only one repeat (genotype A; Table 2). In contrast, all six African M. ulcerans isolates analysed plus strain Agy99 from Ghana had two repeats in all 34 loci (VNTR genotype E; Table 2), indicating that they have further diverged from M. marinum than the other tested geographical lineages of M. ulcerans. The isolates from Australia, Malaysia and Papua New Guinea had one repeat in all 17 subgroup i loci and two repeats in all 15 subgroup ii loci, thus sharing a comparable number of alleles with both the M. marinum-associated VNTR genotype A and the African M. ulcerans-associated genotype E. They could be further subdivided into three VNTR genotypes (designated B–D) by differences in the two VNTR loci 15 and 24 constituting subgroups iii and iv (Table 2). These results indicate an evolutionary scenario in which a progenitor of all M. ulcerans lineages emerged from M. marinum, subsequently diverging into several geographical lineages. The African lineage may have evolved from an ancestral lineage from the Western Pacific region (Australia, Malaysia and Papua New Guinea) which may itself have a progenitor in Asia. These results may confirm the evolutionary scenario suggested by multi-locus sequence typing (Stinear et al., 2000b).
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also described alleles with more than two repeats for 6/9 VNTR loci identified by screening the M. marinum genome. M. marinum genome-assisted identification of new VNTRs has led to the first demonstration of genetic diversity in the same panel of African M. ulcerans isolates as used in this study (Hilty et al., 2006). In contrast, the strain Agy99 genome-based approach used here did not identify another VNTR locus that differentiates African isolates. Therefore, attempts to refine VNTR-based genetic fingerprinting for micro-epidemiological analysis of M. ulcerans should rather focus on the M. marinum than on the M. ulcerans Agy99 genome sequence as a starting point.
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ACKNOWLEDGEMENTS |
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Edited by: G. S. Besra
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 24 November 2006;
revised 16 January 2007;
accepted 17 January 2007.
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