Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study  Jakko van Ingen, PhD, Thomas A Kohl, PhD, Katharina Kranzer, PhD, Barbara Hasse, MD, Peter M Keller, MD, Anna Katarzyna Szafrańska, PhD, Doris Hillemann, PhD, Meera Chand, MBBS, Peter Werner Schreiber, MD, Rami Sommerstein, MD, Christoph Berger, MD, Prof Michele Genoni, MD, Christian Rüegg, MD, Nicolas Troillet, MSc, Andreas F Widmer, MD, Sören L Becker, MD, Prof Mathias Herrmann, MD, Tim Eckmanns, MD, Sebastian Haller, MD, Christiane Höller, MD, Sylvia B Debast, MD, Maurice J Wolfhagen, PhD, Joost Hopman, MD, Prof Jan Kluytmans, PhD, Merel Langelaar, PhD, Daan W Notermans, PhD, Jaap ten Oever, PhD, Peter van den Barselaar, MSc, Alexander B A Vonk, MD, Prof Margreet C Vos, PhD, Nada Ahmed, MSc, Timothy Brown, PhD, Prof Derrick Crook, MBBCh, Theresa Lamagni, PhD, Nick Phin, MD, E Grace Smith, MBChB, Maria Zambon, PhD, Annerose Serr, MD, Tim Götting, MD, Winfried Ebner, MD, Alexander Thürmer, MD, Christian Utpatel, PhD, Cathrin Spröer, PhD, Boyke Bunk, PhD, Ulrich Nübel, PhD, Guido V Bloemberg, PhD, Prof Erik C Böttger, MD, Prof Stefan Niemann, DSc, Dirk Wagner, MD, Prof Hugo Sax, MD  The Lancet Infectious Diseases  Volume 17, Issue 10, Pages 1033-1041 (October 2017) DOI: 10.1016/S1473-3099(17)30324-9 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Alignment of chromosome and plasmid sequences from Mycobacterium chimaera strains ZUERICH-1, ZUERICH-2, DSM 44623, and MC045 The figure was generated using BRIG software.17 (A) The similarity between a central reference chromosome (ZUERICH-1) against query sequences as indicated, and (B) the similarity between reference plasmids (ZUERICH-1, plasmids 1–5) and plasmids from strains ZUERICH-2, DSM 44623, and MC045, respectively. Plasmid 2 (154 kbp) from strain ZUERICH-2 and plasmid 2 (324 kbp) from strain MC045 displayed no homology to any of the plasmids from ZUERICH-1 (not shown). There is no plasmid 1 in MC045, according to NCBI annotation. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 NeighborNet splitstree tree built from 72 545 SNP positions of 250 isolates mapped to the genome of Mycobacterium chimaera DSM 44623 Isolates were grouped with a threshold of maximum 1000 SNPs to the nearest group member, and the four resulting groups are shown. The red line in the tree denotes the division between M chimaera and Mycobacterium intracellulare isolates in the study collection. SNP=single nucleotide polymorphism. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Maximum likelihood tree built from 860 SNP positions of the 200 group 1 isolates mapped to the genome of Mycobacterium chimaera ZUERICH-1 shown as a circular phylogram The maximum SNP distance-based classification, as well as isolate source, country of origin, year of isolation, and major groups or branches are indicated by subsequent coloured circles. Circles on nodes indicate resampling support of at least 90% (green circles) or at least 70% (black circles). Each leaf of the tree is labelled by sample ID and coloured according to its subgroup, with ungrouped samples in white. ECMO=extracorporeal membrane oxygenation. HCU=heater–cooler unit. SNP=single nucleotide polymorphism. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Maximum likelihood tree built from 826 SNP positions of 29 group 2 isolates mapped to the genome of Mycobacterium chimaera ZUERICH-2 shown in circular clade representation and as a rectangular phylogram, with isolate metadata and node support indicated as in figure 2 The leaf colour marks isolates clustered by the threshold of ten SNPs (ie, all that belong to subgroup 2.1). Each leaf of the tree is labelled by sample ID and coloured according to its subgroup, with ungrouped samples in white. SNP=single nucleotide polymorphism. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Relative frequency of isolated strains (n=342; multiple entries for mixed populations) per source and whole-genome sequence group and subgroup attribution The relative strain frequency is depicted as bubble size, showing subgroups 1.1, 1.8, 1.11, and 2.1 in detail, and 1.x (other strains in group 1), 2.x (other strains in group 2), and x (ungrouped strains). Within isolates found to contain mixed populations, each detected strain was included individually. HCU=heater–cooler unit. ECMO=extracorporeal membrane oxygenation. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 The LivaNova 3T heater–cooler unit (A) Side view, opened, revealing the central water reservoir in the upper half of the device. (B) Rear view, with fan grill, tubing, and overflow bucket. The Lancet Infectious Diseases 2017 17, 1033-1041DOI: (10.1016/S1473-3099(17)30324-9) Copyright © 2017 Elsevier Ltd Terms and Conditions