chelonae strain CIP 104535T and M. immunogenum strain CIP 106684T rpoB gene sequences. A heatmap was constructed using the R statistical software based on the spacer

profile as a distance matrix. Results and discussion rpoB identification and rpoB tree The identification of M. abscessus CIP104536T, M. abscessus DSMZ44567, M. bolletii CIP108541T and M. massiliense CIP108297T was confirmed by partial rpoB sequencing. The sequences were deposited in the GenBank database (GenBank accession: KC352778 – KC352795). Isolates P1, P2.1, P2.2, P2.3, P2.4, P2.5, P3.1, P3.2, P4, P5, P6, P7 and P8 exhibited 99% rpoB sequence MG132 similarity with M. abscessus ATCC19977T and were identified as M. abscessus. Isolates P9 NVP-AUY922 mw and P10 exhibited 99% rpoB sequence similarity with “M. bolletii” CIP108541T and were identified as “M. bolletii” whereas isolate P11 exhibited 99% rpoB sequence similarity with “M. massiliense”

CIP108297T and was identified as “M. massiliense”. A total of 23 M. abscessus sequenced genomes were identified as M. abscessus since they exhibited 98 to 100% similarity with the M. abscessus type strain rpoB partial gene sequence. M. abscessus M24 shared 99% similarity with the M. bolletii type strain partial rpoB gene sequence. A total of 26 M. abscessus and “M. massiliense” sequenced genomes shared 99% to 100% similarity with “M. massiliense” partial rpoB gene sequence. The tree built from 69 partial rpoB gene sequences showed three distinct groups, each comprising the type strain (Figure  1a). Figure 1 Phylogenetic tree based on rpoB gene sequence (a); based on the concatenated five MLSA gene sequences (b); and based on the concatenated Methane monooxygenase eight polymorphic spacers (c). Reference MLSA analysis Fragments for the expected size were amplified and sequenced for the five

MLSA genes. The sequences were deposited in the GenBank database (GenBank accession: KC352742 – KC352759, KC352760 – KC352777, KC352796 – KC352813, KC352814 – KC352831, KC352832 – KC352849). Concatenation of the five sequences yielded a total of 19 different types, including 9 types for 37 M. abscessus organisms, four types for 4 “M. bolletii” organisms and M. abscessus M139 and five types for 27 “M. massiliense” organisms. The Hunter-Gaston Index for MLSA was of 0.903. The MLSA tree based on the five gene concatened sequences showed three principal clusters, i.e. a M. abscessus cluster, a “M. bolletii” cluster and a “M. massiliense” cluster (Figure  1b). Latter cluster comprised of five sub-clusters with “M. massiliense” type strain and P11 strain sub-clustering together close to M. abscessus 5S strain. Also, MLSA-derived tree clustered M. abscessus M139 strain and P5 strain respectively identified as “M. massiliense”, close to the “M. bolletii” whereas both strains clustered with M. abscessus in the rpoB gene sequence-derived tree. MST analysis Analysis of the reference M.