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:The Candidatus phylum Omnitrophica (candidate division OP3) occurs ubiquitous in anaerobic habitats, but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiontic cells. In this study, we enriched OP3 cells by double density centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in limonene enrichment cultures, contained empty cells, dead cells and cells devoid of rRNA or both rRNA and DNA according to TEM, thin-section TEM, SEM, CARD-FISH and Live/Dead images. OP3 LiM cells were ultramicrobacteria (200-300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals indicated many rRNA molecules and an active metabolism of OP3 LiM cells attached to Bacteria and to Archaea, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules, an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an RNF complex (Ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin section TEM showed distinctive structures of predation that had been previously observed for “Velamenicoccus”. Our study demonstrated a predatory metabolism for OP3 LiM cells and we propose as name for OP3 LiM Candidatus Velamenicoccus archaeovorus gen. nov., sp. nov..
Project description:Isolation and characterization of two recently isolated Novosphingobium oxfordensis sp. nov. and Novosphingobium mississippiensis sp. nov. strains from soil, with LCMS and genome-based investigation of their glycosphingolipid productions
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