title: Study of the gyrB gene polymorphism as a tool to differentiate among Mycobacterium tuberculosis complex subspecies further underlines the older evolutionary age of 'Mycobacterium canettii'.
abstract: The present investigation evaluated the PCR-restriction fragment length polymorphism (RFLP) analysis of hsp65 and gyrB targets for differentiation of the species within the Mycobacterium tuberculosis complex (MTC) both by including new restriction enzymes and previously unstudied species. The hsp65 restriction analysis using HhaI resulted in a characteristic 'Mycobacterium canettii' pattern. A study of the gyrB gene polymorphism using TaqIalpha and HinfI allowed the initial division of MTC into two major groups, one consisting of M. tuberculosis and 'M. canettii' as opposed to another single group with other species. Three different patterns were observed with RsaI, the first characteristic of Mycobacterium microti, the second with Mycobacterium bovis, M. bovis BCG and Mycobacterium caprae (M. caprae was easily separated from M. bovis, and M. bovis BCG by SacII digestion), and the third with M. tuberculosis, 'M. canettii', Mycobacterium africanum, Mycobacterium pinnipedii, and the dassie bacillus. Although further discrimination within the last group was not obtained using additional restriction enzymes, the HaeIII and RsaI digestions highlighted an important gyrB polymorphism among 'M. canettii' strains. A study of the single nucleotide polymorphisms (SNP) within the gyrB by sequence analysis not only confirmed the results of the restriction analysis, but showed further differences among 'M. canettii' isolates that were not picked up using the existing battery of restriction enzymes. As many as 11 different SNPs were identified in the collection of eight 'M. canettii' isolates studied. Considering that gyrB variability among MTC member species other than 'M. canettii' is as restricted as hsp65 variability among MTC, our data corroborate a recent proposition that the 'M. canettii' group is evolutionary much older than the other MTC members. In conclusion, gyrB PCR-RFLP is a simple and rapid low-cost method that combined with phenotypic characteristics, may be helpful to differentiate most of the subspecies within the MTC.
authors.affiliation: Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Pointe-à-Pitre, Guadeloupe, Morne Jolivière, BP484, F97183 Abymes, Cedex, Guadeloupe.
authors.forename: Khye Seng
authors.initials: KS
authors.lastname: Goh
authors.forename: Michel
authors.initials: M
authors.lastname: Fabre
authors.forename: Richard C
authors.initials: RC
authors.lastname: Huard
authors.forename: Solveig
authors.initials: S
authors.lastname: Schmid
authors.forename: Christophe
authors.initials: C
authors.lastname: Sola
authors.forename: Nalin
authors.initials: N
authors.lastname: Rastogi
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D027081:DNA Gyrase / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
chemical_list: D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D027081:DNA Gyrase