Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains

Abstract

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Fig. 1.

Tree constructed from partial rpoB gene sequences. The tree for all studied strains (n = 123) was generated using the neighbor-joining method. Bootstrap support values (%) are indicated for each node. Species assignment of clinical isolates are according to the criteria in Adékambi et al. (2): ■, M. abscessus; •, M. massiliense; ▴, M. bolletii; numbers in parentheses are the numbers of isolates if there are two or more. Type strains: *, M. abscessus CIP 104536^T; **, M. massiliense CIP 108297^T; ***, M. bolletii CIP 108541^T.

Fig. 2.

Trees constructed from the sequences of the eight individual genes included in the final MLSA scheme. The trees for all studied strains (n = 123) were generated using the neighbor-joining method. Bootstrap support values (%) at each of the nodes are indicated only for trees showing well-defined clusters. Species assignment of clinical isolates according to the criteria of Adékambi et al. (3): ■, M. abscessus; •, M. massiliense; ▴, M. bolletii. Type strains:*, M. abscessus CIP 104536^T; **, M. massiliense CIP 108297^T; ***, M. bolletii CIP 108541^T.

Fig. 3.

Trees constructed from concatenated sequences. (A) Concatenated MLSA sequences. (B) Concatenated MLSA + rpoB sequences. The trees for all studied strains (n = 123) were generated by using the neighbor-joining method. Bootstrap support values (%) are indicated for each node. Each isolate is indicated by its number in our collection. CIP type strains also are indicated. Boxes indicate isolates with discordant rpoB-based identification (see Table 3 for further details). Note that isolates 12 and 63 are located on the M. bolletii branch of the MLSA tree (A) and on the M. massiliense branch of the MLSA + rpoB tree (B).