Downloaded from www.microbiologyresearch.org by IP: 93.91.26.97 On: Wed, 25 Nov 2015 11:30:10 Analysis of the Mycobacterium ulcerans genome sequence reveals new loci for variable number tandem repeats (VNTR) typing Markus Hilty, 1 3 Michael Ka ¨ ser, 1 Jakob Zinsstag, 1 Tim Stinear 2 and Gerd Pluschke 1 Correspondence Markus Hilty m.hilty@imperial.ac.uk 1 Swiss Tropical Institute, 4002 Basel, Switzerland 2 Department of Microbiology, Monash University, Clayton, Victoria, Australia Received 24 November 2006 Revised 16 January 2007 Accepted 17 January 2007 Screening of the genome sequence of the Mycobacterium ulcerans strain Agy99 from Ghana with tandem repeats finder software revealed 34 novel non-degenerate tandem repeats containing loci suitable for variable number tandem repeats (VNTR) typing. All loci revealed polymorphism within M. ulcerans isolates of geographically diverse origins. The results confirm the evolutionary scenario suggested by multi-locus sequence typing in which a progenitor of all M. ulcerans lineages emerged from the environmental species Mycobacterium marinum and subsequently diverged into several geographical lineages. For further attempts to develop a VNTR-based genetic fingerprinting tool for M. ulcerans, it is suggested that the focus should rather be on M. marinum than on the African M. ulcerans Agy99 genome sequence as a starting point. INTRODUCTION Mycobacterium ulcerans,a slow-growing environmental mycobacterium (Yeboah-Manu et al., 2004), 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. tuber- culosis 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. METHODS Identification of the tandem-repeat-containing loci. Tandem repeats finder software (http://tandem.bu.edu/trf/trf.html) was used to screen the M. ulcerans Agy99 sequence database and to identify tandem-repeat-containing loci. Selection criteria were a repeat size of >20 bp, a perfect (100 %) sequence identity of the repeats and an entropy of repeat sequences >1.70 within the total entropy range of 0–2 to allow for easy determination of the number of repeats by agarose gel electrophoresis and to exclude very similar (e.g. GC-rich) sequences which do not represent tandem repeats. 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 informa- tion from the M. marinum genome database (www.sanger.ac.uk/ projects/M_marinum), the M. marinum sequences corresponding to 3Present address: Imperial College, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK. Abbreviation: VNTR, variable number tandem repeats. 2006/004564 G 2007 SGM Printed in Great Britain 1483 Microbiology (2007), 153, 1483–1487 DOI 10.1099/mic.0.2006/004564-0