14 research outputs found
Genomic epidemiology of a major Mycobacterium tuberculosis outbreak: Retrospective cohort study in a low incidence setting using sparse time-series sampling
Substantial molecular evolution and mutation rates in prolonged latent <i>Mycobacterium tuberculosis </i>infection in humans
First outbreak of multi-drug resistant tuberculosis (MDR-TB) in Denmark involving six Danish-born cases
A Major <i>Mycobacterium tuberculosis</i> outbreak caused by one specific genotype in a low-incidence country: Exploring gene profile virulence explanations: Exploring gene profile virulence explanations
Detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary samples with the FluoroType MTBDR assay
Mycobacterium chimaera in Heater–Cooler Units in Denmark Related to Isolates from the United States and United Kingdom
Mycobacterium chimaera was present at high rates (>80%) in heater–cooler units (HCUs) from all 5 thoracic surgery departments in Denmark. Isolates were clonal to HCU-associated isolates from the United States (including some from patients) and United Kingdom. However, M. chimaera from 2 brands of HCU were genetically distinct
Recurrent tuberculosis in patients infected with the predominant Mycobacterium tuberculosis outbreak strain in Denmark. New insights gained through whole genome sequencing
Successful Direct Whole Genome Sequencing and Revivification of Freeze-Dried Nontuberculous Mycobacteria after More than Half a Century of Storage
In this study, 28 “historical” clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting de novo assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. IMPORTANCE The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing