Surveillance and outbreak report Open Access
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Between July and November 2014, 15 community-acquired cases of Legionnaires´ disease (LD), including four with serogroup 1 sequence type (ST) 82, were diagnosed in Northern Zealand, Denmark. An outbreak was suspected. No ST82 isolates were found in environmental samples and no external source was established. Four putative-outbreak ST82 isolates were retrospectively subjected to whole genome sequencing (WGS) followed by phylogenetic analyses with epidemiologically unrelated ST82 sequences. The four putative-outbreak ST82 sequences fell into two clades, the two clades were separated by ca 1,700 single nt polymorphisms (SNP)s when recombination regions were included but only by 12 to 21 SNPs when these were removed. A single putative-outbreak ST82 isolate sequence segregated in the first clade. The other three clustered in the second clade, where all included sequences had < 5 SNP differences between them. Intriguingly, this clade also comprised epidemiologically unrelated isolate sequences from the UK and Denmark dating back as early as 2011. The study confirms that recombination plays a major role in evolution. On the other hand, strains belonging to the same ST can have only few SNP differences despite being sampled over both large timespans and geographic distances. These are two important factors to consider in outbreak investigations.


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  1. Kjelsø C, Espenhain L, Voldstedlund M, Uldum S. Notified cases of legionella pneumonia in 2014. EPI-NEWS. 2015; 44. Available from: http://www.ssi.dk/English/News/EPI-NEWS/2015/No%2044%20-%202015.aspx
  2. European Commission. Commission Implementing Decision of 8 August 2012 amending Decision 2002/253/EC laying down case definitions for reporting communicable diseases to the Community network under Decision No 2119/98/EC of the European Parliament and of the Council (2012/506/EU). Official Journal of the European Union. Luxembourg: Publications Office of the European Union; 27.9.2012:L262. Available from: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2012:262:0001:0057:EN:PDF
  3. Reischl U, Linde HJ, Lehn N, Landt O, Barratt K, Wellinghausen N. Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis. J Clin Microbiol. 2002;40(10):3814-7.  https://doi.org/10.1128/JCM.40.10.3814-3817.2002  PMID: 12354888 
  4. Helbig JH, Kurtz JB, Pastoris MC, Pelaz C, Lück PC. Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups. J Clin Microbiol. 1997;35(11):2841-5. PMID: 9350744 
  5. Helbig JH, Bernander S, Castellani Pastoris M, Etienne J, Gaia V, Lauwers S, et al. Pan-European study on culture-proven Legionnaires’ disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur J Clin Microbiol Infect Dis. 2002;21(10):710-6.  https://doi.org/10.1007/s10096-002-0820-3  PMID: 12415469 
  6. Gaia V, Fry NK, Afshar B, Lück PC, Meugnier H, Etienne J, et al. Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila. J Clin Microbiol. 2005;43(5):2047-52.  https://doi.org/10.1128/JCM.43.5.2047-2052.2005  PMID: 15872220 
  7. Ratzow S, Gaia V, Helbig JH, Fry NK, Lück PC. Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J Clin Microbiol. 2007;45(6):1965-8.  https://doi.org/10.1128/JCM.00261-07  PMID: 17409215 
  8. Ginevra C, Lopez M, Forey F, Reyrolle M, Meugnier H, Vandenesch F, et al. Evaluation of a nested-PCR-derived sequence-based typing method applied directly to respiratory samples from patients with Legionnaires’ disease. J Clin Microbiol. 2009;47(4):981-7.  https://doi.org/10.1128/JCM.02071-08  PMID: 19225096 
  9. Mérault N, Rusniok C, Jarraud S, Gomez-Valero L, Cazalet C, Marin M, et al. DELPH-I Study Group. Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples. Appl Environ Microbiol. 2011;77(5):1708-17.  https://doi.org/10.1128/AEM.02261-10  PMID: 21193672 
  10. Mentasti M, Afshar B, Collins S, Walker J, Harrison TG, Chalker V. Rapid investigation of cases and clusters of Legionnaires’ disease in England and Wales using direct molecular typing. J Med Microbiol. 2016;65(6):484-93.  https://doi.org/10.1099/jmm.0.000257  PMID: 27046155 
  11. Mentasti M, Cassier P, David S, Ginevra C, Gomez-Valero L, Underwood A, et al. ESCMID Study Group for Legionella Infections (ESGLI). Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47. Clin Microbiol Infect. 2017;23(4):264.e1-9.  https://doi.org/10.1016/j.cmi.2016.11.019  PMID: 27915212 
  12. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754-60.  https://doi.org/10.1093/bioinformatics/btp324  PMID: 19451168 
  13. Delcher AL, Phillippy A, Carlton J, Salzberg SL. Fast algorithms for large-scale genome alignment and comparison. Nucleic Acids Res. 2002;30(11):2478-83.  https://doi.org/10.1093/nar/30.11.2478  PMID: 12034836 
  14. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, et al. Versatile and open software for comparing large genomes. Genome Biol. 2004;5(2):R12.  https://doi.org/10.1186/gb-2004-5-2-r12  PMID: 14759262 
  15. Croucher NJ, Page AJ, Connor TR, Delaney AJ, Keane JA, Bentley SD, et al. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015;43(3):e15.  https://doi.org/10.1093/nar/gku1196  PMID: 25414349 
  16. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-9.  https://doi.org/10.1093/bioinformatics/btu153  PMID: 24642063 
  17. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31(22):3691-3.  https://doi.org/10.1093/bioinformatics/btv421  PMID: 26198102 
  18. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-77.  https://doi.org/10.1089/cmb.2012.0021  PMID: 22506599 
  19. Schalk JA, Euser SM, van Heijnsbergen E, Bruin JP, den Boer JW, de Roda Husman AM. Soil as a source of Legionella pneumophila sequence type 47. Int J Infect Dis. 2014;27:18-9.  https://doi.org/10.1016/j.ijid.2014.05.009  PMID: 25130616 
  20. García-Fulgueiras A, Navarro C, Fenoll D, García J, González-Diego P, Jiménez-Buñuales T, et al. Legionnaires’ disease outbreak in Murcia, Spain. Emerg Infect Dis. 2003;9(8):915-21.  https://doi.org/10.3201/eid0908.030337  PMID: 12967487 
  21. Nguyen TM, Ilef D, Jarraud S, Rouil L, Campese C, Che D, et al. A community-wide outbreak of legionnaires disease linked to industrial cooling towers--how far can contaminated aerosols spread? J Infect Dis. 2006;193(1):102-11.  https://doi.org/10.1086/498575  PMID: 16323138 
  22. Borgen K, Aaberge I, Werner-Johansen O, Gjøsund K, Størsrud B, Haugsten S, et al. A cluster of Legionnaires disease linked to an industrial plant in southeast Norway, June-July 2008. Euro Surveill. 2008;13(38):18985. PMID: 18801321 
  23. Herwaldt LA, Gorman GW, McGrath T, Toma S, Brake B, Hightower AW, et al. A new Legionella species, Legionella feeleii species nova, causes Pontiac fever in an automobile plant. Ann Intern Med. 1984;100(3):333-8.  https://doi.org/10.7326/0003-4819-100-3-333  PMID: 6696354 
  24. Muraca PW, Stout JE, Yu VL, Yee YC. Legionnaires’ disease in the work environment: implications for environmental health. Am Ind Hyg Assoc J. 1988;49(11):584-90.  https://doi.org/10.1080/15298668891380268  PMID: 3195473 
  25. Ricketts KD, Joseph C, Lee J, Wewalka GEuropean Working Group for Legionella Infections. Survey on legislation regarding wet cooling systems in European countries. Euro Surveill. 2008;13(38):18982. PMID: 18801320 
  26. Sánchez-Busó L, Comas I, Jorques G, González-Candelas F. Recombination drives genome evolution in outbreak-related Legionella pneumophila isolates. Nat Genet. 2014;46(11):1205-11.  https://doi.org/10.1038/ng.3114  PMID: 25282102 
  27. McAdam PR, Vander Broek CW, Lindsay DS, Ward MJ, Hanson MF, Gillies M, et al. Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires’ disease outbreak. Genome Biol. 2014;15(11):504. PMID: 25370747 
  28. Raphael BH, Baker DJ, Nazarian E, Lapierre P, Bopp D, Kozak-Muiznieks NA, et al. Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State. Appl Environ Microbiol. 2016;82(12):3582-90.  https://doi.org/10.1128/AEM.00362-16  PMID: 27060122 
  29. Reuter S, Harrison TG, Köser CU, Ellington MJ, Smith GP, Parkhill J, et al. A pilot study of rapid whole-genome sequencing for the investigation of a Legionella outbreak. BMJ Open. 2013;3(1):e002175.  https://doi.org/10.1136/bmjopen-2012-002175  PMID: 23306006 
  30. Graham RM, Doyle CJ, Jennison AV. Real-time investigation of a Legionella pneumophila outbreak using whole genome sequencing. Epidemiol Infect. 2014;142(11):2347-51.  https://doi.org/10.1017/S0950268814000375  PMID: 24576553 
  31. Underwood AP, Jones G, Mentasti M, Fry NK, Harrison TG. Comparison of the Legionella pneumophila population structure as determined by sequence-based typing and whole genome sequencing. BMC Microbiol. 2013;13(1):302.  https://doi.org/10.1186/1471-2180-13-302  PMID: 24364868 
  32. Coscollá M, Fernández C, Colomina J, Sánchez-Busó L, González-Candelas F. Mixed infection by Legionella pneumophila in outbreak patients. Int J Med Microbiol. 2014;304(3-4):307-13.  https://doi.org/10.1016/j.ijmm.2013.11.002  PMID: 24309206 
  33. David S, Rusniok C, Mentasti M, Gomez-Valero L, Harris SR, Lechat P, et al. Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently. Genome Res. 2016;26(11):1555-64. PMID: 27662900 
  34. Mercante JW, Morrison SS, Desai HP, Raphael BH, Winchell JM. Genomic Analysis Reveals Novel Diversity among the 1976 Philadelphia Legionnaires' Disease Outbreak Isolates and Additional ST36 Strains. PLoS One. 2016 Sep 29;11(9):e0164074. doi:  https://doi.org/10.1371/journal.pone.0164074 . eCollection 2016.
  35. David S, Mentasti M, Tewolde R, Aslett M, Harris SR, Afshar B, et al. Evaluation of an optimal epidemiologic typing scheme for Legionella pneumophila with whole genome sequence data using validation guidelines. J Clin Microbiol. 2016;54(8):2135-48. PMID: 27280420 

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