Microbial Ecology

, Volume 68, Issue 1, pp 121–131 | Cite as

Integration and Proliferation of Pseudomonas aeruginosa PA01 in Multispecies Biofilms

  • Mahtab Ghadakpour
  • Elanna Bester
  • Steven N. Liss
  • Michael Gardam
  • Ian Droppo
  • S. Hota
  • Gideon M. Wolfaardt
Environmental Microbiology


Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.



The research was funded by grants from the Canada Research Chair Program and the Natural Sciences and Engineering Research Council of Canada.


  1. 1.
    Bester E, Wolfaardt G, Joubert L, Garny K, Saftic S (2005) Planktonic-cell yield of a Pseudomonad biofilm. Appl Environ Microbiol 71:7792–7798PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Bester E, Edwards EA, Wolfaardt GM (2009) Planktonic cell yield is linked to biofilm development. Can J Microbiol 55:1195–1206CrossRefPubMedGoogle Scholar
  3. 3.
    Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Wingender J, Flemming H (2011) Biofilms in drinking water and their role as reservoir for pathogens. Int J Hyg Environ Health 214:417–423CrossRefPubMedGoogle Scholar
  5. 5.
    Breathnach AS, Cubbon MD, Karunaharan RN, Pope CF, Planche TD (2012) Multidrug-resistant Pseudomonas aeruginosa outbreaks in two hospitals: association with contaminated hospital waste-water systems. J Hosp Infect 82:19–24CrossRefPubMedGoogle Scholar
  6. 6.
    Hota S, Hirji Z, Stockton K, Lemieux C, Dedier H, Wolfaardt G, Gardam MA (2009) Outbreak of multidrug-resistant Pseudomonas aeruginosa colonization and infection secondary to imperfect intensive care unit room design. Infect Control Hosp Epidemiol 30:25–33CrossRefPubMedGoogle Scholar
  7. 7.
    Yapicioglu H, Gokmen TG, Yildizdas D, Koksal F, Ozlu F, Kale-Cekinmez E, Mert K, Mutlu B, Satar M, Narli N, Candevir A (2012) Pseudomonas aeruginosa infections due to electronic faucets in a neonatal intensive care unit. J Paediatr Child Health 48:430–434CrossRefPubMedGoogle Scholar
  8. 8.
    Gilbert P, Maira-Litran T, McBain AJ, Rickard AH, Whyte FW (2002) The physiology and collective recalcitrance of microbial biofilm communities. Adv Microb Physiol 46:203–256CrossRefGoogle Scholar
  9. 9.
    Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322CrossRefPubMedGoogle Scholar
  10. 10.
    Hall-Stoodley L, Stoodley P (2005) Biofilm formation and dispersal and the transmission of human pathogens. Trends Microbiol 13:7–10CrossRefPubMedGoogle Scholar
  11. 11.
    Anaissie EJ, Penzak SR, Dignani MC (2002) The hospital water supply as a source of nosocomial infections. Arch Intern Med 162:1483–1492CrossRefPubMedGoogle Scholar
  12. 12.
    Pajkos A, Vickery K, Cossart Y (2004) Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination? J Hosp Infect 58:224–229CrossRefPubMedGoogle Scholar
  13. 13.
    Walker JT, Bradshaw DJ, Finney M, Fulford MR, Frandsen E, Ostergaard E, Ten Cate JM, Moorer WR, Schel AJ, Mavridou A, Kamma JJ, Mandilara G, Stosser L, Kneist S, Araujo R, Contreras N, Goroncy-Bermes P, O'Mullane D, Burke F, Forde A, O'Sullivan M, Marsh PD (2004) Microbiological evaluation of dental unit water systems in general dental practice in Europe. Eur J Oral Sci 112:412–418CrossRefPubMedGoogle Scholar
  14. 14.
    World Health Organization (2011) Report on the burden of endemic health care-associated infection worldwide. A systematic review of the literature. ISBN 978 92 4 150150 7Google Scholar
  15. 15.
    Lambertsen L, Sternberg C, Molin S (2004) Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol 6:726–732CrossRefPubMedGoogle Scholar
  16. 16.
    Wolfaardt GM, Lawrence JR, Robarts RD, Caldwell SJ, Caldwell DE (1994) Multicellular organization in a degradative biofilm community. Appl Environ Microb 60:434–446Google Scholar
  17. 17.
    Bester E, Kroukamp O, Wolfaardt GM, Boonzaaier L, Liss SN (2010) Metabolic differentiation in biofilms as indicated by carbon dioxide production rates. Appl Environ Microbiol 76:1189–1197PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Lewandowski Z, Beyenal H, Stookey D (2004) Reproducibility of biofilm processes and the meaning of steady state in biofilm reactors. Water Sci Technol 49:359–364PubMedGoogle Scholar
  19. 19.
    Lewis K (2008) Multidrug tolerance of biofilms and persister cells. Bacterial Biofilms 322:107–131CrossRefGoogle Scholar
  20. 20.
    Chen X, Stewart PS (2000) Biofilm removal caused by chemical treatments. Water Res 34:4229–4233CrossRefGoogle Scholar
  21. 21.
    Stewart PS (2002) Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol 292:107–113CrossRefPubMedGoogle Scholar
  22. 22.
    Altman SJ, McGrath LK, Souza CA, Murton JK, Camper AK (2009) Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms. J Appl Microbiol 107:287–299CrossRefPubMedGoogle Scholar
  23. 23.
    Moritz MM, Flemming HC, 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
  24. 24.
    Stoodley P, Sauer K, Davies DG, Costerton JW (2002) Biofilms as complex differentiated communities. Annu Rev Microbiol 56:187–209CrossRefPubMedGoogle Scholar
  25. 25.
    Purevdorj-Gage B, Costerton WJ, Stoodley P (2005) Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms. Microbiology-Sgm 151:1569–1576CrossRefGoogle Scholar
  26. 26.
    Kirov SM, Webb JS, O'May CY, Reid DW, Woo JKK, Rice SA, Kjelleberg S (2007) Biofilm differentiation and dispersal in mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Microbiology-Sgm 153:3264–3274CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mahtab Ghadakpour
    • 1
  • Elanna Bester
    • 1
  • Steven N. Liss
    • 2
  • Michael Gardam
    • 3
    • 4
  • Ian Droppo
    • 5
  • S. Hota
    • 3
    • 4
  • Gideon M. Wolfaardt
    • 1
    • 6
  1. 1.Department of Chemistry and BiologyRyerson UniversityTorontoCanada
  2. 2.School of Environmental StudiesQueen’s UniversityKingstonCanada
  3. 3.Infection Prevention and ControlUniversity Health NetworkTorontoCanada
  4. 4.Department of MedicineUniversity of TorontoTorontoCanada
  5. 5.Water Science and Technology DirectorateEnvironment CanadaBurlingtonCanada
  6. 6.Stellenbosch Institute for Advanced StudyWallenberg Research Centre at Stellenbosch UniversityStellenboschSouth Africa

Personalised recommendations