Investigation of Heterotrophic Bacteria, Legionella and Free-Living Amoeba in Cooling Tower Samples by FISH and Culture Methods

Year 2017, Volume: 76 Issue: 1, 7 - 13, 01.06.2017

Zuhal Zeybek , Nihal Dogruoz Gungor İrfan Turetgen

https://doi.org/10.5152/EurJBiol.2017.1702

Abstract

The
microorganisms living in the cooling towers water can affect both human health
through inhalation of aerosolized water as well as industrial processes. In
order to analyse such man-made water systems, microbiological tests that can
give results in a short time are needed. In this study, the presence of
heterotrophic bacteria, Legionella bacteria and free - living amoeba, FLA,
including Acanthamoeba, in cooling-tower water and biofilm samples were
investigated using two different methods, fluorescent in situ hybridization
(FISH), and culture. For this, a total of 40 water and biofilm samples were
taken from 16 different cooling towers in Istanbul. FISH and culture analysis
have revealed that the number of heterotrophic bacteria within the water and
the biofilm samples was above the threshold values (>105 cell. mL-1),
generally. Despite Acanthamoeba were present in all cooling tower specimens,
Legionella pneumophila serogroup 1 were only detected in the biofilm of one
cooling tower. According to the results of this study, both methods are
recommended to be used in conjunction. Due to the the large biodiversity of FLA
such as Hartmanella sp. and Naeglaria sp, there is a need for new studies
utilizing FISH method for sensitive, reliable results in a short period of
time.

Keywords

Cooling tower water, biofilm, heterotrophic bacteria, Legionella, Acanthamoeba, free-living amoebae

References

• 1. Kimiran Erdem A, Sanli Yurudu NO, Arslan Aydogdu EO, Dogruoz Gungor N, Zeybek Z, Turetgen I, et al. Quantitative microbiological analysis of biofilm communities from the surfaces of different cooling tower materials. IUFS J Biol 2008; 67(1): 9-16. 2. Minnos B, Ilhan Sungur E, Cotuk A, Dogruoz Gungor N, Cansever N. The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor. Biofouling 2013; 29 (3): 223-35. 3. Samimi A. Microorganisms of cooling tower problems and how to manage Them. Int J Basic Appl Sci 2013; 1: 705-15. 4. Sanlı Yurudu NO. Sogutma kulesi dezenfeksiyonunda is sagligi ve guvenligi. Tes Müh 2015a; 146: 13-23. 5. Turetgen I, Sungur EI, Cotuk A. Enumeration of Legionella pneumophila in cooling tower water systems. Environ Monit Assess 2005; 100(1-3): 53-8. 6. Miller RD, Kenepp KA. Risk Assesments for Legionnaires Disease based on routine surveillance of cooling towers for Legionellae. In: Barbaree, J.M., Breiman, R.F., Dufour, R.F(eds.), Legionella Current Status and Emerging Perspectives, ISBN 1-55581-055-1, 40-43, ASM, USA, 1993. 7. South Australian Public Health Regulations https://www.legislation.sa.gov.au/LZ/C/R/SOUTH%20AUSTRALIAN%20PUBLIC%20HEALTH%20(LEGIONELLA)%20REGULATIONS%202013/CURRENT/2013.42.UN.PDF (2017, March 29) 2013. 8. Nalco, Australian Standards Maintaining Cooling Water Systems, http://www.ihea.org.au/files/sa/maintenance_of_cooling_water_systems.pdf (2017, March 14) 2015. 9. Sanli Yurudu NO. Lejyoner Hastalıgı Kontrol Usul ve Esasları Hakkinda Yonetmelik” ile Ilgili Bilgilendirme. Tes Muh 2015; 149: 33-36. 10. Kuiper MW, Wullings B, Akkermans ADL, Beumer RR, Kooij D. Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl Environ Microbiol 2004; 70(11): 6826-33. 11. Storey MV, Ashbolt NJ, Stenstrom TA. Biofilms, thermophilic amoebae and Legionella pneumophila - a quantitative risk assessment for distributed water. Water Sci Technol 2004; 50(1): 77-82. 12. Barbeau J, Buchler T. Biofilm augment the number of free-living amoebae in dental unit waterlines. Res Microbiol 2001; 152(8): 753-60. 13. Declerck P, Behets J, Lammertyn E, Ollevıer F. Acanthamoeba castellanii strongly increases the number of Legionella pneumophila in model tap water biofilms. In: Cianciotto, N., Kwaik, Y. A, Edelstein, P., Fields, B., Geary, D., Harrison, T., Joseph, C., Ratcliff, R. M., Stout, J. E., Swanson, M. S. (eds), Legionella: State of the Art 30 Years after Its Recognition, 395- 397, Washington, D.C. 2006. 14. Horn M, Wagner M. Bacterial endosymbionts of FLA. J Eukaryot Microbiol 2004; 51(5): 3-16. 15. Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y. Amoeba as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 2005; 71(1): 20-8. 16. Rowbotham TJ. Preliminary report on the pathogenity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 1980; 33(12): 1179-83. 17. Rodriguez-Zaragoza S. Ecology of free-living amoebae. Crit Rev Microbiol 1994; 20(3): 225-41. 18. Scheikl U, Sommer R, Kirschner A, Rameder A, Schrammel B, Zwimuller I, et al. Free-living amoeba (FLA) co-occuring with legionella in Industrial waters . Eur J Prositol 2014; 50(4): 422-9. 19. Scheikl U, Tsao HF, Horn M, Indra A, Walochnik J. Free-living amoeba and their associated bacteria in Austrian cooling towers: A 1 year routine screening. Parasitol Res 2016; 115(9): 3364-75. 20. Thomas JM, Ashbolt NJ. Do free-living amoebae in treated drinking water systems present an emerging health risk. Environ Sci Technol 2011; 45(3): 860-9. 21. Huck PM, Gagnon G. Understanding the distribution system as a bioreactor: a framework for managing heterotrophic plate count levels. Int J Food Microbiol 2004; 92(3): 347-53. 22. Reasoner DJ, Geldrich EE. A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 1985; 49(1): 1-7. 23. British Standard. Water quality part 4: Microbiological methods, detection and enumeration of Legionella. BS 6068-4.12:2004, ISO 11731. 1998. 24. National Standard Method. Isolation and identification of Acanthamoeba species. Issued by standards unit, evaluations, and standards laboratory on behalf of the water working group and the environmental surveillance unit, CDSC, W 17i2.3, 1-12. 2005 25. Grimm D, Merkert H, Ludwig W, Schleifer, KH, Hacker J, Brand BC. Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe. Appl Environ Microbiol 1998; 64(7): 2686-90. 26. Grimm D, Ludwig W, Brandt BC, Mıchel R, Schleıfer KH, Hacker J, Steınert M. Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella - positive environmental samples. Syst Appl Microbiol 2001; 24(1): 76-82. 27. Manz W, Amann R, Szewzyk R, Szewzyk U, Stenstrom TA, Hutzler P, et al. In situ identification of Legionellaceae using16S rRNA targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology 1995; 141(Pt 1): 29-39. 28. Gescher DM, Kovacevic D, Schmiedel D, Siemoneit S, Mallmann C, Halle E, et al. Fluorescence in situ hybridisation (FISH) accelerates identification of Gram-positive cocci in positive blood cultures. Int J Antimicrob Agents 2008; 32S: S51–S59. 29. Stothard DR, Hay J, Schroeder-Dıedrıch JM, Seal DV, Byers TJ. Fluorescent oligonucleotide probes for clinical and environmental detection of Acanthamoeba and the T4 18S rRNA gene sequence type. J Clin Microbiol 1999; 37(8): 2687-93. 30. Wallner G, Amann R, and Beisker W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 1993; 14(2): 136-43. 31. Gauthier MJ. Environmental parameters associated with the viable but nonculturable state. In: R. Colwell and D. Jay Grimes (eds.), Nonculturable microorganisms in the environment, Chapter 7, 87-112, ASM Press, Washington DC. 2000. 32. Cotuk A, Dogruoz Gungor N, Zeybek Z, Kimiran Erdem A, Ilhan Sungur E. The effects of Pseudomonas and Aeromonas strains on Legionella pneumophila growth. Ann Microbiol 2005; 55(3): 219-24. 33. Kimura S, Tateda K, Ishii Y, Horikawa M, Miyairi S, Gotoh N, et al. Pseudomonas aeruginosa Las quorumsensing autoinducer suppresses growth and biofilm production in Legionella species. Microbiology 2009; 155(Pt 6): 1934-9. 34. Smirnow AV, Brown S. Guide to the methods of study and identification of soil gymnamoebae. Protistology 2004; 3(3): 148-90.