Environmental Science and Pollution Research

, Volume 22, Issue 10, pp 7628–7640 | Cite as

Environmental surveillance and molecular epidemiology of waterborne pathogen Legionella pneumophila in health-care facilities of Northeastern Greece: a 4-year survey

  • Ioanna G. Alexandropoulou
  • Spyridon Ntougias
  • Theocharis G. Konstantinidis
  • Theodoros A. Parasidis
  • Maria Panopoulou
  • Theodoros C. Constantinidis
Research Article


A 4-year proactive environmental surveillance of Legionella spp. in the water distribution and cooling systems of five health-care facilities was carried out as part of the strategy for the prevention of hospital-acquired Legionnaires’ disease in Northeastern Greece. Legionella spp. were detected in 71 out of 458 collected samples. The majority of strains belonged to Legionella pneumophila serogroups 2–15 (75.0 %), while all L. pneumophila serogroup 1 strains (23.6 %) were isolated from a single hospital. The highest percentage of positive samples was found in distal sites (19.4 %), while no Legionella strains were detected in cooling systems. Each hospital was colonized at least once with L. pneumophila, while remedial actions resulted in significant reduction of Legionella concentration. The molecular epidemiology of environmental L. pneumophila strains was also investigated using random amplified polymorphic DNA (RAPD) and multi-gene sequence-based analysis. Based on RAPD patterns, L. pneumophila serogroups 2–15 and serogroup 1 strains were classified into 24 and 9 operational taxonomic units (OTUs), respectively. Sequencing of housekeeping and diversifying pressure-related genes recommended by European Working Group for Legionella Infections (EWGLI) revealed not only a high intraspecies variability but also the circulation and persistence of one specific genotyping profile in the majority of hospitals. This study highlights the necessity for diachronic surveillance of Legionella in health-care facilities by adopting both cultural and molecular methods.


Waterborne pathogens Environmental epidemiology Environmental monitoring Legionella diversity Water distribution systems Hospitals acquired legionellosis 



The authors greatly appreciate Dr. Norman Fry for authorizing access to the EWGLI SBT database. They also like to thank the environmental health officers and the hospitals’ infection committee of Northeastern Greece for their cooperation during the study period.

Supplementary material

11356_2014_3740_MOESM1_ESM.doc (96 kb)
ESM 1 (DOC 96 kb)


  1. Arvand M, Jungkind K, Hack A (2011) Contamination of the cold water distribution system of health care facilities by Legionella pneumophila: do we know the true dimension? Euro Surveill 16Google Scholar
  2. Bansal NS, Mc Donell F (1997) Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis. J Clin Microbiol 35:2310–2314Google Scholar
  3. Beauté J, Zucs P, de Jong B (2013) Legionnaires’ disease in Europe, 2009–2010. Euro Surveill 18Google Scholar
  4. Borella P, Montagna MT, Stampi S, Stancanelli G, Romano-Spica V, Triassi M et al (2005) Legionella contamination in hot water of Italian hotels. Appl Environ Microbiol 71:5805–5813CrossRefGoogle Scholar
  5. Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Etienne J et al (2008) Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. Genome Res 18:431–441CrossRefGoogle Scholar
  6. Doleans A, Aurell H, Reyrolle M, Lina G, Freney J, Vandenesch F et al (2004) Clinical and environmental distributions of Legionella strains in France are different. J Clin Microbiol 42:458–460CrossRefGoogle Scholar
  7. Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15:506–526CrossRefGoogle Scholar
  8. Francisque A, Rodriguez MJ, Miranda-Moreno LF, Sadiq R, Proulx F (2009) Modeling of heterotrophic bacteria counts in a water distribution system. Water Res 43:1075–1087CrossRefGoogle Scholar
  9. Gaia V, Fry NK, Afshar B, Lück PC, Meugnier H, Etienne J et al (2005) A consensus sequence-based epidemiological typing scheme for clinical and environmental isolates of Legionella pneumophila. J Clin Microbiol 43:2047–2052CrossRefGoogle Scholar
  10. Garcia-Nuñez M, Sopena N, Ragull S, Pedro-Botet ML, Morera J, Sabria M (2008) Persistence of Legionella in hospital water supplies and nosocomial Legionnaires’ disease. FEMS Immunol Med Microbiol 52:202–206CrossRefGoogle Scholar
  11. Gouy M, Guindon S, Gascuel O (2010) SeaView Version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224CrossRefGoogle Scholar
  12. Kimura S, Tateda K, Ishii Y, Horikawa M, Miyairi S, Gotoh N, Ishiguro M, Yamaguchi K (2009) Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species. Microbiology 155:1934–1939CrossRefGoogle Scholar
  13. Ko KS, Lee HK, Park MY, Park MS, Lee KH, Woo SY et al (2002) Population genetic structure of Legionella pneumophila inferred from RNA polymerase gene (rpoB) and DotA gene (dotA) sequences. J Bacteriol 184:2123–2130CrossRefGoogle Scholar
  14. Lam MC, Ang LW, Tan AL, James L, Goh KT (2011) Epidemiology and control of legionellosis. Singapore Emerg Infect Dis 17:1209–1215CrossRefGoogle Scholar
  15. Leoni E, De Luca G, Legnani PP, Sacchetti R, Stampi S, Zanetti F (2005) Legionella waterline colonization: detection of Legionella species in domestic, hotel and hospital hot water systems. J Appl Microbiol 98:373–379CrossRefGoogle Scholar
  16. Lin YE, Stout JE, Yu VL (2011) Prevention of hospital-acquired legionellosis. Curr Opin Infect Dis 24:350–356CrossRefGoogle Scholar
  17. Mentasti M, Underwood A, Lück C, Kozak-Muiznieks NA, Harrison TG, Fry NK (2013) Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene. Clin Microbiol Infect. doi: 10.1111/1469-0691.12459 Google Scholar
  18. Neil K, Berkelman R (2008) Increasing incidence of legionellosis in the United States, 1990–2005: changing epidemiologic trends. Clin Infect Dis 47:591–599CrossRefGoogle Scholar
  19. Ozerol IH, Bayraktar M, Cizmeci Z, Durmaz R, Akbas E, Yildirim Z et al (2006) Legionnaire’s disease: a nosocomial outbreak in Turkey. J Hosp Infect 62:50–57CrossRefGoogle Scholar
  20. Ratcliff RM, Lanser JA, Manning PA, Heuzenroeder MW (1998) Sequence-based classification scheme for the genus Legionella targeting the mip gene. J Clin Microbiol 36:1560–1567Google Scholar
  21. Ratzow S, Gaia V, Helbig JH, Fry NK, Luck PC (2007) 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 45:1965–1968CrossRefGoogle Scholar
  22. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, van Dijl JM, Laurent F, et al. (2013) Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 18Google Scholar
  23. Sabrià M, Mòdol JM, Garcia-Nuñez M, Reynaga E, Pedro-Botet ML, Sopena N et al (2004) Environmental cultures and hospital-acquired Legionnaires’ disease: a 5-year prospective study in 20 hospitals in Catalonia, Spain. Infect Control Hosp Epidemiol 25:1072–1076CrossRefGoogle Scholar
  24. Sabria M, Yu VL (2002) Hospital-acquired legionellosis: solutions for a preventable infection. Lancet Infect Dis 2:368–373CrossRefGoogle Scholar
  25. Serrano-Suárez A, Dellundé J, Salvadó H, Cervero-Aragó S, Méndez J, Canals O, Blanco S, Arcas A, Araujo R (2013) Microbial and physicochemical parameters associated with Legionella contamination in hot water recirculation systems. Environ Sci Poll Res 20:5534–5544CrossRefGoogle Scholar
  26. Tan MJ, Tan JS, File TM Jr (2002) Legionnaires disease with bacteremic coinfection. Clin Infect Dis 35:533–539CrossRefGoogle Scholar
  27. Tesauro M, Bianchi A, Consonni M, Pregliasco F, Galli MG (2010) Environmental surveillance of Legionella pneumophila in two Italian hospitals. Ann Ist Super Sanita 46:274–278Google Scholar
  28. Van de Peer Y, De Wachter R (1993) TREECON: a software package for the construction and drawing of evolutionary trees. Comput Appl Biosci 9:177–182Google Scholar
  29. Velonakis E, Karanika M, Mouchtouri V, Thanasias E, Katsiaflaka A, Vatopoulos A et al (2012) Decreasing trend of Legionella isolation in a long-term microbial monitoring program in Greek hospitals. Int J Environ Health Res 22:197–209CrossRefGoogle Scholar
  30. von Baum H, Ewig S, Marre R, Suttorp N, Gonschior S, Welte T et al (2008) Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis 46:1356–1364CrossRefGoogle Scholar
  31. Ulleryd P, Hugosson A, Allestam G, Bernander S, Claesson BE, Eilertz I et al (2012) Legionnaires’ disease from a cooling tower in a community outbreak in Lidköping, Sweden—epidemiological, environmental and microbiological investigation supported by meteorological modelling. BMC Infect Dis 12:313CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ioanna G. Alexandropoulou
    • 1
  • Spyridon Ntougias
    • 2
  • Theocharis G. Konstantinidis
    • 1
  • Theodoros A. Parasidis
    • 1
  • Maria Panopoulou
    • 3
  • Theodoros C. Constantinidis
    • 1
  1. 1.Laboratory of Hygiene and Environmental Protection, Medical SchoolDemocritus University of ThraceAlexandroupolisGreece
  2. 2.Department of Environmental EngineeringDemocritus University of ThraceXanthiGreece
  3. 3.Microbiology Laboratory, Medical SchoolDemocritus University of ThraceAlexandroupolisGreece

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