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Frozen in Time: Unlocking the History of Nontuberculous Mycobacteria

X ES Iversen(1) A Norman(1) D B Folkvardsen(1) E Svensson(1) E M Rasmussen(1) T Lillebaek(1,2)

1:International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark; 2:Global Health Section, Department of Public Health, University of Copenhagen, Denmark

The global incidence and prevalence of nontuberculous mycobacteria (NTM) disease and colonization continue to rise. In a pilot study, we explored our unique collection of freeze-dried mycobacterial strains stored at the International Reference Laboratory of Mycobacteriology (IRLM) at Statens Serum Institut (SSI), Copenhagen, since 1948. We selected 28 ‘historical’ NTM isolates collected from patients between 1948 to 1957. We investigated their viability using whole genome sequencing (WGS) on DNA extracted directly from a suspension of freeze-dried cells versus after culturing. The DNA quality was evaluated by analyzing the per-base quality scores of paired-end sequencing reads and the overall contiguity of resulting de novo assemblies. Remarkably, all isolates had remained viable after seven decades in storage, as they were easily re-cultured. No DNA degradation was observed when analyzing sequence data from the freeze-dried cells. These findings emphasize the value of freeze-drying for long-term storage and demonstrate that sequencing directly on freeze-dried mycobacteria without prior re-cultivation can save laboratory time and resources. Furthermore, analysis of historical isolates revealed four previously unknown Mycobacterium strains isolated from patients between 1948 to 1955.
NTM infection and disease remain poorly studied and many aspects are still unknown, a field that needs to be improved. Our study lays the groundwork for future investigations of the IRLM strain collection, which comprises approximately 4,000 isolates. Furthermore, ongoing studies aim to enhance our understanding of NTM by exploring the course of epidemiology, etiology, and resistance mechanisms of NTM over decades.

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