Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus

Abstract

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

Fig 1. Neighbor-joining phylogenetic trees based on the partial hsp65 and rpoB genes of 6 Rec-mass strains.

Phylogenetic trees of 6 Rec-mas strains based on (A) the partial hsp65 gene (603 bp) and (B) the partial rpoB gene (711 bp) sequences. All the trees were constructed using the neighbor-joining method in the MEGA 4.0 program. The bootstrap values were calculated from 1,000 replications and values <50% were not shown. Black-centered circles indicate that the corresponding clusters were supported with maximum parsimony-based trees. The bar indicates the number of base substitutions per site. Black-centered triangles indicate that the corresponding sequences were sequenced and obtained in this study.

Fig 2. Neighbor-joining phylogenetic trees based on the 7 MLST genes of 6 Rec-mass strains.

Phylogenetic trees of 6 Rec-mas strains from the partial sequencing of seven housekeeping genes. (A) argH, (B) cya, (C) glpK, (D) gnd, (E) murC, (F) pta and (G) purH gene sequence based trees were constructed by using the neighbor-joining method in the MEGA 4.0 program. The bootstrap values were calculated from 1,000 replications and values <50% were not shown. Black-centered circles indicate that the corresponding clusters were supported with maximum parsimony-based trees. The bar indicates the number of base substitutions per site. Black-centered triangles indicate that the corresponding sequences were sequenced and obtained in this study.

Fig 3. Neighbor-joining phylogenetic trees of 6 Rec-mas strains from concatenated sequences.

The phylogenetic tree based on (A) concatenated 7 MLST gene sequences, (B) concatenated 7 MLST genes and hsp65 gene sequences, and (C) concatenated 7 MLST genes, hsp65 and rpoB gene sequences. The trees for all studied strains were generated by using the neighbor-joining method. Bootstrap support values (%) from 1,000 replications are indicated for each node and values <50% were not shown. Black centered circles indicate that the corresponding clusters were supported with maximum parsimony-based trees. The bar indicates the number of base substitutions per site.

Fig 4. The identification of 6 Rec-mas strains into subspecies levels by PCR targeting the erm(41) gene.

M, 1 kb DNA ladder; Lane 1, M. abscessus CIP 104536^T; Lane 2, M. massiliense CIP 108297^T; Lane 3, M. bolletii CIP 108541^T; Lane 4, Asan 50594 (Type II); Lane 5, Asan 50375 (Type I, Seq2); Lane 6, Asan 52748 (Type I, Seq1); Lane 7, Asan 53996 (Type I, Seq1); Lane 8, Asan 54142 (Type I, Seq1); Lane 9, Asan 56120 (Type I, Seq1); Lane 10, Asan 61912 (Type I, Seq1); N, negative control.

Fig 5. Recombination analysis of Rec-mas strains in the complete rpoB gene sequence.

(A) A BootScan Plot was constructed based on a pairwise distance model in the RDP4 program. A BootScan support percent of over 70% (cutoff value, dotted line) was considered significant. The green line represents the comparison of rpoB sequences from M. abscessus and M. massiliense Type strains. The blue line represents the comparison of rpoB sequences from M. massiliense Type and Rec-mas (Asan 50375 and 54142) strains. The yellow line represents the comparison of rpoB sequences from M. abscessus Type and Rec-mas (Asan 50375 and 54142) strains. The estimated breakpoints are tagged on the schematic diagram of the Rec-mas rpoB gene. (B) Neighbor-joining trees based on the rpoB sequences excluding the 478 bp recombination region (blue) and only the recombination region (yellow).