Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping

Abstract

Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS 6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex.

FIG. 1.

Approximate locations in Europe of M. caprae isolates from animals (filled circles) or humans (open circles) included in the study. The exact provenance was unavailable for nine human isolates from Germany (shown boxed).

FIG. 2.

Neighbor-joining tree based on 12 MIRU-VNTR loci and derived from 166 MTC isolates (76 M. caprae isolates from this study and 90 non-M. caprae MTC isolates taken from Supply et al. [45]). The out-group taxon for tree reconstruction was M. canettii. The linkage distance scale is indicated at the bottom. M. caprae isolates are indicated by the gray bar at the right and designated by country code of origin and RI number (see list in supplemental material). Other MTC isolates are designated by country code followed by the isolate number, as in reference . The three isolates printed in bold and marked with an asterisk are mentioned in the text.