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: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.
Project description:A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium isolates obtained from a variety of host species. DNA microarrays were used as a platform for comparing mycobacteria field isolates with the sequenced bovine isolate Mycobacterium avium subsp. paratuberculosis (Map) K10. ORFs were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to Map K10 DNA. Map isolates cultured from cattle, bison, sheep, goat, avian, and human sources were hybridized to the Map microarray. Three large deletions were observed in the genomes of four Map isolates obtained from sheep and four clusters of ORFs homologous to sequences in the Mycobacterium avium subsp. avium (Maa) 104 genome were identified as being present in these isolates. One of these clusters encodes glycopeptidolipid biosynthesis enzymes. One of the Map sheep isolates had a genome profile similar to a group of Mycobacterium avium subsp. silvaticum (Mas) isolates which included four independent laboratory stocks of the organism traditionally identified as Maa strain 18. Genome diversity in Map appears to be mostly restricted to large sequence polymorphisms that are often associated with mobile genetic elements. Keywords: Comparative genomic hybridization
Project description:A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium isolates obtained from a variety of host species. DNA microarrays were used as a platform for comparing mycobacteria field isolates with the sequenced bovine isolate Mycobacterium avium subsp. paratuberculosis (Map) K10. ORFs were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to Map K10 DNA. Map isolates cultured from cattle, bison, sheep, goat, avian, and human sources were hybridized to the Map microarray. Three large deletions were observed in the genomes of four Map isolates obtained from sheep and four clusters of ORFs homologous to sequences in the Mycobacterium avium subsp. avium (Maa) 104 genome were identified as being present in these isolates. One of these clusters encodes glycopeptidolipid biosynthesis enzymes. One of the Map sheep isolates had a genome profile similar to a group of Mycobacterium avium subsp. silvaticum (Mas) isolates which included four independent laboratory stocks of the organism traditionally identified as Maa strain 18. Genome diversity in Map appears to be mostly restricted to large sequence polymorphisms that are often associated with mobile genetic elements. Keywords: Comparative genomic hybridization Each isolate was competitively hybridized against Map K10 with a minimum of 2 dye flip hybridizations per isolate.
Project description:Mycobacterium abscessus is an opportunistic pathogen notorious for its resistance to most classes of antibiotics and low cure rates. In addition to the highly impermeable mycomembrane, M. abscessus carries an array of shared and species-specific defence mechanisms. However, it remains unknown whether M. abscessus’ antibiotic stress response is fine-tuned or an all-or-nothing response. A deeper understanding of underlying resistance and tolerance mechanisms is pivotal in development of targeted therapeutic regimens. We elucidate the transcriptomic response of M. abscessus to antibiotics recommended in treatment guidelines. The M. abscessus ATCC 19977 strain was used. Bacteria were subjected to sub-inhibitory concentrations of drugs for 4- and 24-hours, followed by RNA sequencing. In addition, time-kill kinetic analysis was performed using bacteria after pre-exposure to clarithromycin, amikacin or tigecycline for 24-hours. Lastly, Pan-genome analysis of 35 strains from all three subspecies was performed. Mycobacterium abscessus shows both drug-specific and communal transcriptomic responses to antibiotic exposure. Key features of its tolerance to antibiotics are drug-specific converting enzymes, target protection and shifts in its respiratory chain and metabolic state. The observed transcriptomic responses are likely not strain-specific, as genes involved in tolerance are found in all included strains, with the exception of erm(41) in M. abscessus subspecies massiliense. Due to the communal response elicited by ribosomal-targeting antibiotics, exposure to any of these drugs rapidly induces tolerance mechanisms that decrease susceptibility to ribosome-targeting drugs from multiple classes. Screening high-risk patients (e.g. those with bronchiectasis) for M. abscessus infection prior to starting macrolide or aminoglycoside maintenance therapy is warranted.
Project description:Members of the Mycobacterium (M.) abscessus complex (MABC) are rapidly growing mycobacteria showing smooth and/or rough colony morphotype. While not as virulent as M. tuberculosis, they can cause soft tissue infection and fatal pulmonary disease, especially in patients with cystic fibrosis. Diagnosing MABC pulmonary disease is challenging since the isolation of M. abscessus from respiratory samples is in itself not diagnostic and the clinical features are often non-specific. Immunologic assays, which could aid in the understanding and diagnosis of the disease, are not available. In this study eight rough and six smooth colony morphotype isolates were collected from seven clinical MABC strains and the M. abscessus reference strain ATCC19977, as six strains showed both morphotypes simultaneously and two strains only showed a rough morphotype. Clinical isolates were submitted to whole genome sequencing. Quantitative proteomic analysis was performed on bacterial lysates and the culture supernatant of all 14 isolates. Supernatant proteins present in all isolates were compared in a BLAST search against other clinically significant mycobacterial species to determine species-specific proteins of MABC. In silico B- and T-cell epitope prediction was performed for species-specific proteins. All clinical strains were found to be M. abscessus ssp. abscessus. Six of seven rough colony clinical isolates contained genetic changes in the MAB_4099c gene, which is a likely genetic basis for the rough morphotype. Proteomic analysis detected 3 137 different proteins in total of which 79 proteins were found in the culture supernatants of all isolates. BLAST analyses of these 79 proteins identified 12 of those exclusively encoded by all members of MABC plus M. immunogenum. In silico prediction of epitopes predicted B- and T-cell epitopes in all these 12 species-specific proteins, rendering them promising candidates for future studies on immune pathogenesis and immune diagnostic tools for MABC disease.