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Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

ABSTRACT: The molecular typing methods used so far for Mycobacterium ulcerans isolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity among M. ulcerans isolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1. Three different genotypes were found in Ghana, demonstrating for the first time the genetic diversity of M. ulcerans in an African country. While the ST1/MIRU 1 allele combination BD/BAA seems to dominate in Africa, it was only rarely found in isolates from Ghana, where the combination BD/B was dominant and observed in all districts studied. A third variant genotype (C/BAA) was found only in the Amansie-West district. The results indicate that new genetic variants of M. ulcerans emerged and spread within Ghana and support the potential of VNTR-based typing for genotyping of M. ulcerans.

SUBMITTER: Hilty M

PROVIDER: S-EPMC1367230 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

Hilty Markus M Yeboah-Manu Dorothy D Boakye Daniel D Mensah-Quainoo Ernestina E Rondini Simona S Schelling Esther E Ofori-Adjei David D Portaels Françoise F Zinsstag Jakob J Pluschke Gerd G

Journal of bacteriology 20060201 4

The molecular typing methods used so far for Mycobacterium ulcerans isolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity among M. ulcerans isolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously describe ...[more]

PMID: 16452429

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Project description:BackgroundCurrently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.ResultsAn eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.ConclusionThis paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

Project description:BackgroundThe genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.ResultsGenotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.ConclusionIn a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Dataset Information

Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

Publications

Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

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