Abstract
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.
Highlights
Samples were collected from all the heater–cooler units (HCUs) located in thoracic surgery departments (n = 5) throughout the country
If the test result was positive for M. intracellulare, we performed internal transcribed spacer (ITS) sequencing [1,2]
Initial PCR for ITS sequencing was done with a 5 μL sample of DNA in a 50 μL reaction containing 5 μL 10× PCR buffer, 10 μL 5× Q-solution, 2 μL 25 mM MgCl2, 1 μL 10 μM primer ITS_F, 1 μL 10 μM primer ITS_R [1,2], 0.25 μL HotStarTaq (HotStarTaq Master Mix Kit; QIAGEN, Hilden, Germany), and water (DNase, RNase, and protease free)
Summary
The frozen aliquots were thawed and diluted with 100 mL of phosphate buffer and passed through a MicroFunnel 300 black membrane filter with a 0.45-μm pore size (Pall Corporation, Westborough, MA, USA). The sequencing was performed using the Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. 1 mL of a culture enriched in Dubos broth (SSI Diagnostika, Hilleroed, Denmark) was centrifuged at 13,000 rpm for 10 min, the supernatant removed, and the pellet resuspended in 300 μL TE buffer (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Paired-end Illumina reads were end-trimmed (base-quality >Q3; minimum end-trimmed length >35 bp) and filtered using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic), and mapped to the 5.8 Mbp M. chimera FI01069 chromosome sequence
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