8371 0.6038 0.8084 0.7158 0.912 a MST = Multispacer Sequence Typing. b isolates were listed with reference to their corresponding patient, for example P1 = isolate 1 from patient 1, P2.1 = isolate 1 from patient 2, etc. c DI = Discrimination index. MST based tree and comparaison with rpoB identification and MLSA analysis The MST-phylogenetic tree clustered
isolates from patients P1 to P8 with M. abscessus reference strain, isolates from P9 and P10 with “M. bolletii” and isolate from P11 with “M. massiliense”, in agreement with their rpoB sequence-based identification and MLSA analysis (Figure click here 1c). The MST, MLSA and rpoB phylogenetic trees separated the M. abscessus isolates into three principal clusters depicted by M. abscessus, “M. bolletii” and “M. massiliense” isolates (Figure 1a, b and c). However, MST resolved “M. bolletii” cluster into two sub-clusters formed by isolate P5 and all of the other M. bolletii isolates with a 76% bootstrap value, wich is discordant with MLSA and rpoB based tree.
Each cluster or sub-cluster of the M. abscessus isolates corresponded to different genotypes. The “M. massiliense” Androgen Receptor inhibitor cluster was more disperse and divided into six sub-clusters with isolate P11 and “M. massiliense” type strain sub-clustering alone. The results of this analysis were consistent for 67 isolates and inconsistent for two isolates P5 and M. abscessus M139. A heatmap incorporating all spacer patterns into a matrix further demonstrated that spacer n°2 was the most discriminating spacer (Figure 2). Hence, the tree based on the spacer n°2 sequence also discriminated the three M. abscessus, “M. bolletii” and “M. massiliense” clusters (Figure 3). This discrimination potential makes spacer n°2 a useful new tool for the accurate identification of M. abscessus subspecies. Furthermore, these data indicated that it was readily possible to discriminate isolates that would have been identified as “M. bolletii”  or “M. massiliense”  using a previous taxonomy proposal and are now grouped as M. abscessus subsp. bolletii according
to a recent taxonomy proposal [20, 21]. Figure 2 Heatmap and clustering of M. abscessus mycobacteria under study based in difference of profile. Figure 3 Phylogenetic Cisplatin tree based on MST spacer n°2 sequence. Conclusion We herein developed a sequencing-based MST genotyping technique that allows the accurate identification and discrimination of M. abscessus mycobacteria. Therefore, MST could be added to the panel of molecular methods currently available for genotyping M. abscessus mycobacteria, with the advantages that MST is a PCR and sequencing-based technique, thereby providing a robust and accurate result without requiring a high DNA concentration and purity, as is the case for pulsed-field gel electrophoresis (PFGE)  and randomly amplified polymorphic DNA (RAPD) .