Project description:Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus group] comprises three closely related taxa with taxonomic status under revision: M. abscessus sensu stricto, M. bolletii and M. massiliense. We describe here a simple, robust and cost effective PCR-based method for distinguishing among M. abscessus, M. massiliense and bolletii. Based on the M. abscessus ATCC 19977T genome, discriminatory regions were identified between M. abscessus and M. massiliense from array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense and 2 M. bolletii previously identified by multi-target sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full length erm(41) instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for a straightforward identification of M. abscessus, M. massiliense and M. bolletii and assessment of inducible clarithromycin resistance. This method can be easily implemented into a routine workflow providing subspecies level identification within 24 hours of isolation of M. abscessus group. Two-color CGH with 4 independent Mycobacterium clinical isolates and the M massiliense type strain (CCUG 48898) labeled with Cy3 were cohybridized with the M abscessus type strain (ATCC 19977) labeled with Cy5 on a tiling array designed against the M abscessus type strain
Project description:It has been reported that lateral gene transfer (LGT) events among Mycobacteroides abscessus strains are prevalent. The hsp65 gene, a chronometer gene for bacterial phylogenetic analysis, is resistant to LGT events, particularly among mycobacterial strains, rendering the hsp65-targeting method the most widely used method for mycobacterial detection. To determine the prevalence of M. abscessus strains that are subject to hsp65 LGT, we applied rpoB typing to 100 clinically isolated Korean strains of M. abscessus that had been identified by hsp65 sequence analysis. The analysis indicated the presence of 2 rough strains, showing a discrepancy between the 2 typing methods. MLST analysis based on the partial sequencing of seven housekeeping genes, erm(41) PCR and further hsp65 PCR-restriction enzyme and polymorphism analysis (PRA) were conducted to identify the two strains. The MLST results showed that the two strains belong to M. abscessus subsp. massiliense and not to M. abscessus subsp. abscessus, as indicated by the rpoB-based analysis, suggesting that their hsp65 genes are subject to LGT from M. abscessus subsp. abscessus. Further analysis of these strains using the hsp65 PRA method indicated that these strains possess a PRA pattern identical to that of M. abscessus subsp. abscessus and distinct from that of M. abscessus subsp. massiliense. In conclusion, we identified two M. abscessus subsp. massiliense rough strains from Korean patients with hsp65 genes that might be laterally transferred from M. abscessus subsp. abscessus. To the best of our knowledge, this is the first demonstration of possible LGT events associated with the hsp65 gene in mycobacteria. Our results also suggest that there is the potential for misidentification when the hsp65-based protocol is used for mycobacterial identification.
Project description:Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus group] comprises three closely related taxa with taxonomic status under revision: M. abscessus sensu stricto, M. bolletii and M. massiliense. We describe here a simple, robust and cost effective PCR-based method for distinguishing among M. abscessus, M. massiliense and bolletii. Based on the M. abscessus ATCC 19977T genome, discriminatory regions were identified between M. abscessus and M. massiliense from array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense and 2 M. bolletii previously identified by multi-target sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full length erm(41) instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for a straightforward identification of M. abscessus, M. massiliense and M. bolletii and assessment of inducible clarithromycin resistance. This method can be easily implemented into a routine workflow providing subspecies level identification within 24 hours of isolation of M. abscessus group.