Differentiation among members of the Mycobacterium tuberculosis complex by molecular and biochemical features: evidence for two pyrazinamide-susceptible subtypes of M. bovis.

The variations in biochemical as well as molecular characteristics among several members of the Mycobacterium tuberculosis complex that are not M. tuberculosis have been assessed to facilitate an unambiguous species identification. Altogether, 96 M. tuberculosis complex strains including 52 M. bovis isolates and 44 M. africanum isolates were analyzed by spoligotyping. The strains could be clustered into five spoligotype groups. All M. bovis isolates showed the typical absence of the spacers 39 to 43 and typical biochemical properties. However, within these strains we found a group of strains that had a spoligotype pattern which is clearly defined by the additional absence of spacers 3 to 16 and that were uncommonly susceptible to pyrazinamide (PZA). This spoligotype pattern has previously been described as being typical for a caprine genotype because of its predominant isolation from sheep and goats. Due to the clinical importance of PZA resistance, we propose two M. bovis subtypes: M. bovis subtype bovis, which is resistant to PZA, and M. bovis subtype caprae, which is susceptible to PZA. Two additional strains that clustered in group 3 showed biochemical and genetic properties typical for M. bovis and were also sensitive to PZA; thus, they may represent a third PZA-susceptible M. bovis subtype. The M. africanum isolates could be clustered into two spoligotype groups which can be differentiated from M. bovis by hybridization to spacers 39 to 43. These groups correspond to the previously described M. africanum subtypes I and II and can be clearly distinguished from each other by spoligotyping and resistance to thiophen-2-carboxylic acid hydrazide. Our results demonstrate that spoligotyping is a useful tool for differentiation of M. bovis and M. africanum. Moreover, we describe two PZA-susceptible M. bovis subtypes and describe a method that facilitates an unambiguous differentiation of the two M. africanum subtypes.