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.

Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

“…bolletii. Recently developed schemes for multispacer sequence typing (MST) (20) and multilocus sequence analysis (MLSA) (21) confirmed the existence of three principal groups within M. abscessus sensu lato, with each containing one of the type strains (M. abscessus CIP 104536, M. massiliense CIP 108297, and M. bolletii CIP 108541). However, several reports have described isolates showing ambiguous identification by partial sequencing of different DNA targets (7,10,(22)(23)(24)(25), suggesting the occurrence of genetic exchange among members of the M. abscessus group and indicating that the taxonomy of M. abscessus sensu lato is far from being resolved.…”

Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

“…massiliense, and M. abscessus subsp. bolletii (18)(19)(20)(21). This delineation is important because clinical studies suggest differing treatment outcomes for patients infected with M. abscessus subsp.…”

Evaluation of the GenoType® NTM DR for Subspecies Identification and Determination of Drug Resistance in Clinical M. abscessus Isolates

“…massiliense (susceptible on days 3 and 14) and an erm(41) sequence showing the characteristic deletions of nucleotides 64-65 and 159-432. This has been described by others [20] and reflects the uncertainty of subspecies differentiation for M. abscessus when using a single gene like rpoB. Here, WGS can be of advantage as it allows more accurate assessment of an individual isolate in comparison to reference genomes of all three M. abscessus subspecies.…”