, Volume 28, Issue 2, pp 329–339 | Cite as

Influence of copper surfaces on biofilm formation by Legionella pneumophila in potable water

  • M. S. GiãoEmail author
  • S. A. Wilks
  • C. W. Keevil


Legionella pneumophila is a waterborne pathogen that can cause Legionnaires’ disease, a fatal pneumonia, or Pontiac fever, a mild form of disease. Copper is an antimicrobial material used for thousands of years. Its incorporation in several surface materials to control the transmission of pathogens has been gaining importance in the past decade. In this work, the ability of copper to control the survival of L. pneumophila in biofilms was studied. For that, the incorporation of L. pneumophila in polymicrobial drinking water biofilms formed on copper, PVC and PEX, and L. pneumophila mono-species biofilms formed on copper and uPVC were studied by comparing cultivable and total numbers (quantified by peptide nucleic acid (PNA) hybridisation). L. pneumophila was never recovered by culture from heterotrophic biofilms; however, PNA-positive numbers were slightly higher in biofilms formed on copper (5.9 × 105 cells cm−2) than on PVC (2.8 × 105 cells cm−2) and PEX (1.7 × 105 cells cm−2). L. pneumophila mono-species biofilms grown on copper gave 6.9 × 105 cells cm−2 for PNA-positive cells and 4.8 × 105 CFU cm−2 for cultivable numbers, showing that copper is not directly effective in killing L. pneumophila. Therefore previous published studies showing inactivation of L. pneumophila by copper surfaces in potable water polymicrobial species biofilms must be carefully interpreted.


Legionella pneumophila Drinking water biofilms PNA hybridisation Copper 



This research was supported by the Copper Development Association, New York, NY, and the International Copper Association, New York, NY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


  1. Atlas RM (1999) Legionella: from environmental habitats to disease pathology, detection and control. Environ Microbiol 1:283–293CrossRefPubMedGoogle Scholar
  2. Azevedo NF, Vieira MJ, Keevil CW (2003) Development of peptide nucleic acid probes to detect Helicobacter pylori in diverse species potable water biofilms. In: McBain A, Allison C, Brading M, Rickard A, Verran J, Walker J (eds) Biofilm communities: order from chaos? Bioline, Cardiff, pp 231–239Google Scholar
  3. Bleichert P, Espírito Santo C, Hanczaruk M, Meyer H, Grass G (2014) Inactivation of bacterial and viral biothreat agents on metallic copper surfaces. Biometals 27:1179–1189CrossRefPubMedGoogle Scholar
  4. Borkow G, Gabbay J (2009) Copper, an ancient remedy returning to fight microbial, fungal and viral infections. Curr Chem Biol 3:272–278Google Scholar
  5. Buse HY, Lu J, Struewing IT, Ashbolt NJ (2014) Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons. Int J Hyg Environ Health 217:219–225CrossRefPubMedGoogle Scholar
  6. Casey AL, Adams D, Karpanen TJ et al (2010) Role of copper in reducing hospital environment contamination. J Hosp Infect 74:72–77CrossRefPubMedGoogle Scholar
  7. Declerck P (2010) Biofilms: the environmental playground of Legionella pneumophila. Environ Microbiol 12:557–566CrossRefPubMedGoogle Scholar
  8. Gião MS, Azevedo NF, Wilks SA, Vieira MJ, Keevil CW (2008) Persistence of Helicobacter pylori in heterotrophic drinking water biofilms. Appl Environ Microbiol 74:5898–5904CrossRefPubMedCentralPubMedGoogle Scholar
  9. Gião M, Wilks S, Azevedo N, Vieira M, Keevil C (2009a) Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila. Microb Ecol 58:56–62CrossRefPubMedGoogle Scholar
  10. Gião MS, Wilks S, Azevedo NF, Vieira MJ, Keevil CW (2009b) Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress. Biofouling 25:345–351CrossRefPubMedGoogle Scholar
  11. Gião MS, Wilks SA, Azevedo NF, Vieira MJ, Keevil CW (2009c) Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms. Biofouling 25:335–343CrossRefPubMedGoogle Scholar
  12. Gião MS, Azevedo NF, Wilks SA, Vieira MJ, Keevil CW (2011) Interaction of Legionella pneumophila and Helicobacter pylori with bacterial species isolated from drinking water biofilms. BMC Microbiol 11:57CrossRefPubMedCentralPubMedGoogle Scholar
  13. Gould S, Fielder M, Kelly A, Morgan M, Kenny J, Naughton D (2009) The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens. Ann Microbiol 59:151–156CrossRefGoogle Scholar
  14. Guerrieri E, Bondi M, Sabia C, de Niederhäusern S, Borella P, Messi P (2008) Effect of bacterial interference on biofilm development by Legionella pneumophila. Curr Microbiol 57:532–536CrossRefPubMedGoogle Scholar
  15. Hussong D, Colwell RR, O’Brien M, Weiss E, Pearson AD, Weiner RM, Burge WD (1987) Viable Legionella pneumophila not detectable by culture on agar media. Nat Biotechnol 5:947–950CrossRefGoogle Scholar
  16. James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R (1999) Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low nutrient environments. Appl Environ Microbiol 65:822–827PubMedCentralPubMedGoogle Scholar
  17. Keevil CW (2001) Continuous culture models to study pathogens in biofilms. Method Enzymol 337:104–122CrossRefGoogle Scholar
  18. Keevil CW (2002) Pathogens in environmental biofilms. In: Bitton G (ed) The encyclopedia of environmental microbiology. Wiley, New York, pp 2339–2356Google Scholar
  19. Keevil CW (2003) Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy. Water Sci Technol 47:105–116PubMedGoogle Scholar
  20. Landeen LK, Yahya MT, Gerba CP (1989) Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila. Appl Environ Microbiol 55:3045–3050PubMedCentralPubMedGoogle Scholar
  21. Lehtola MJ, Torvinen E, Miettinen LT, Keevil CW (2006) Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable water biofilms. Appl Environ Microbiol 72:848–853CrossRefPubMedCentralPubMedGoogle Scholar
  22. Lin YSE, Vidic RD, Stout JE, Yu VL (2002) Negative effect of high pH on biocidal efficacy of copper and silver ions in controlling Legionella pneumophila. Appl Environ Microbiol 68:2711–2715CrossRefPubMedCentralPubMedGoogle Scholar
  23. Lu H, Clarke M (2005) Dynamic properties of Legionella containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007CrossRefPubMedGoogle Scholar
  24. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR (1977) Legionnaires’ disease—isolation of a bacterium and demonstration of its role in other respiratory disease. New Engl J Med 297:1197–1203CrossRefPubMedGoogle Scholar
  25. Moritz MM, Flemming H-C, Wingender J (2010) Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials. Int J Hyg Environ Health 213:190–197CrossRefPubMedGoogle Scholar
  26. Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, Donlan RM (2001) Role of biofilms in the survival of Legionella pneumophila in a model potable water system. Microbiol 147:3121–3126Google Scholar
  27. Nieto JJ, Fernández-Castillo R, Márquez MC, Ventosa A, Quesada E, Ruiz-Berraquero F (1989) Survey of metal tolerance in moderately halophilic eubacteria. Appl Environ Microbiol 55:2385–2390PubMedCentralPubMedGoogle Scholar
  28. Noyce JO, Michels H, Keevil CW (2006) Use of copper cast alloys to control Escherichia coli O157 cross-contamination during food processing. Appl Environ Microbiol 72:4239–4244CrossRefPubMedCentralPubMedGoogle Scholar
  29. Pasculle W (2000) Update on Legionella. Clin Microbiol Newsl 22:97–101CrossRefGoogle Scholar
  30. Rogers J, Dowsett AB, Dennis PJ, Lee JV, Keevil CW (1994a) Influence of plumbing materials on biofilm formation and growth of Legionella pneumophila in potable water systems. Appl Environ Microbiol 60:1842–1851PubMedCentralPubMedGoogle Scholar
  31. Rogers J, Dowsett AB, Dennis PJ, Lee JV, Keevil CW (1994b) Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora. Appl Environ Microbiol 60:1585–1592PubMedCentralPubMedGoogle Scholar
  32. Salgado CDMD, Sepkowitz KAMD, John JFMD et al (2013) Copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit. Infect Control Hosp Epidemiol 34:479–486CrossRefPubMedGoogle Scholar
  33. Schmidt M, Attaway H, Terzieva S et al (2012a) Characterization and control of the microbial community affiliated with copper or aluminum heat exchangers of HVAC systems. Curr Microbiol 65:141–149CrossRefPubMedCentralPubMedGoogle Scholar
  34. Schmidt MG, Attaway HH, Sharpe PA et al (2012b) Sustained reduction of microbial burden on common hospital surfaces through introduction of copper. J Clin Microbiol 50:2217–2223CrossRefPubMedCentralPubMedGoogle Scholar
  35. Surman SB, Morton LHG, Keevil CW (1994) The dependence of Legionella pneumophila on other aquatic bacteria for survival on R2A medium. Int Biodeterior Biodegrad 13:223–236CrossRefGoogle Scholar
  36. Surman S, Morton G, Keevil B, Fitzgeorge R (2002) Legionella pneumophila proliferation is not dependent on intracellular replication. In: Marre R et al (eds) Legionella. ASM Press, Washingtom DC, pp 86–89Google Scholar
  37. Türetgen I, Cotuk A (2007) Monitoring of biofilm-associated Legionella pneumophila on different substrata in model cooling tower system. Environ Monit Assess 125:271–279CrossRefPubMedGoogle Scholar
  38. van der Kooij D, Veenendaal HR, Scheffer WJH (2005) Biofilm formation and multiplication of Legionella in a model warm water system with pipes of copper, stainless steel and cross-linked polyethylene. Water Res 39:2789–2798CrossRefPubMedGoogle Scholar
  39. Wadowsky RM, Yee RB (1983) Satellite growth of Legionella pneumophila with an environmental isolate of Flavobacterium breve. Appl Environ Microbiol 46:1447–1449PubMedCentralPubMedGoogle Scholar
  40. Walker JT, Sonesson A, Keevil CW, White DC (1993) Detection of Legionella pneumophila in biofilms containing a complex microbial consortium by gas chromatography-mass spectrometry analysis of genus-specific hydroxy fatty acids. FEMS Microbiol Lett 113:139–144CrossRefPubMedGoogle Scholar
  41. Warnes SL, Keevil CW (2013) Inactivation of norovirus on dry copper alloy surfaces. PLoS One 8:e75017. doi: 10.1371/journal.pone.0075017 CrossRefPubMedCentralPubMedGoogle Scholar
  42. Warnes SL, Caves V, Keevil CW (2012) Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for gram-positive bacteria. Environ Microbiol 14:1730–1743CrossRefPubMedGoogle Scholar
  43. Warnes SL, Summersgill EN, Keevil CW (2014) Inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity. Appl Environ Microbiol 81(3):1085–1091CrossRefPubMedCentralPubMedGoogle Scholar
  44. Wilks SA, Keevil CW (2006) Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of legionellae in biofilms. Appl Environ Microbiol 72:5453–5462CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Environmental Healthcare Unit, Centre for Biological Sciences, Life Sciences Building, Highfield CampusUniversity of SouthamptonSouthamptonUK

Personalised recommendations