Abstract
Wastewater treatment plants are environmental niches for Legionella pneumophila, the most commonly identified causative agent of severe pneumonia known as Legionnaire’s disease. In the present study, Legionella pneumophila’s concentrations were monitored in an industrial wastewater treatment plant and environmental isolates were characterized concerning their growth kinetics with respect to temperature and their inhibition by organic acids and ammonium. The results of the monitoring study showed that Legionella pneumophila occurs in activated sludge tanks operated with very different sludge retention times, 2.5 days in a complete-mix reactor, and 10 days in a membrane bioreactor, indicating that this bacterium can grow at different rates, despite the same wastewater temperature of 35 °C. The morphology of Legionella cells is different in both reactors; in the membrane bioreactor, the bacteria grow in clusters, while in the complete-mix reactor, filaments predominate demonstrating a faster growth rate. Legionella pneumophila concentrations in the complete-mix reactor and in the membrane bioreactor were within the range 3 × 101 to 4.8 × 103 GU/mL and 3 × 102 to 4.7 × 103 GU/mL, respectively. Environmental Legionella pneumophila SG2–14 isolates showed distinct temperature preferences. The lowest growth rate was observed at 28 °C, and the highest 0.34 d−1 was obtained at 42 °C. The presence of high concentrations of organic acids and ammonium found in anaerobically pre-treated wastewater caused growth inhibition. Despite the increasing research efforts, the mechanisms governing the growth of Legionella pneumophila in wastewater treatment plants are still unclear. New innovative strategies to prevent the proliferation of this bacterium in wastewater are in demand.
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References
Allestam G, de Jong B, Långmark J (2006) Legionella. American Society of Microbiology, Washington, D.C. https://doi.org/10.1128/9781555815660
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
Borella P, Guerrieri E, Marchesi I, Bondi M, Messi P (2005) Water ecology of Legionella and protozoan: environmental and public health perspectives. Biotechnol Annu Rev 11:355–380. https://doi.org/10.1016/S1387-2656(05)11011-4
Buse HY, Ashbolt NJ (2011) Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing. Lett Appl Microbiol 53:217–224. https://doi.org/10.1111/j.1472-765X.2011.03094.x
Buse HY, Schoen ME, Ashbolt NJ (2012) Legionellae in engineered systems and use of quantitative microbial risk assessment to predict exposure. Water Res 46:921–933. https://doi.org/10.1016/j.watres.2011.12.022
Bushell FML, Tonner PD, Jabbari S, Schmid AK, Lund PA (2019) Synergistic impacts of organic acids and pH on growth of Pseudomonas aeruginosa: a comparison of parametric and bayesian non-parametric methods to model growth. Front Microbiol 9:3196. https://doi.org/10.3389/fmicb.2018.03196
Caicedo C, Beutel S, Scheper T, Rosenwinkel KH, Nogueira R (2016) Occurrence of Legionella in wastewater treatment plants linked to wastewater characteristics. Environ Sci Pollut Res Int 23:16873–16881. https://doi.org/10.1007/s11356-016-7090-6
Caicedo C, Rosenwinkel K-H, Nogueira R (2018) Temperature-driven growth of Legionella in lab-scale activated sludge systems and interaction with protozoa. Int J Hyg Environ Health 221:315–322. https://doi.org/10.1016/J.IJHEH.2017.12.003
Caicedo C, Rosenwinkel K-H, Exner M, Verstraete W, Suchenwirth R, Hartemann P, Nogueira R (2019) Legionella occurrence in municipal and industrial wastewater treatment plants and risks of reclaimed wastewater reuse: review. Water Res 149:21–34. https://doi.org/10.1016/J.WATRES.2018.10.080
ECDC (2015) E.C. for D.P. and C. Annual epidemiological report for 2015. Stockholm
Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15:506–526. https://doi.org/10.1128/CMR.15.3.506-526.2002
Fliermans CB, Cherry WB, Orrison LH, Smith SJ, Tison DL, Pope DH (1981) Ecological distribution of Legionella pneumophila. Appl Environ Microbiol 41:9–16
Fykse EM, Aarskaug T, Thrane I, Blatny JM (2013) Legionella and non-Legionella bacteria in a biological treatment plant. Can J Microbiol 59:102–109. https://doi.org/10.1139/cjm-2012-0166
Grimm D (2000) Development and evaluation of novel detection systems specific for legionellae and amoebae and their application in ecological studies. Doctoral thesis, Faculty of Biology University of Würzburg
Grimm D, Merkert H, Ludwig W, Schleifer KH, Hacker J, Brand BC (1998) Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe. Appl Environ Microbiol 64:2686–2690
Justice SS, Hunstad DA, Cegelski L, Hultgren SJ (2008) Morphological plasticity as a bacterial survival strategy. Nat Rev Microbiol 6:162–168. https://doi.org/10.1038/nrmicro1820
Kahm M, Hasenbrink G, Lichtenberg-Fraté H, Ludwig J, Kschischo M (2010) Grofit: fitting biological growth curves with R. J Stat Softw 33:1–21. https://doi.org/10.18637/jss.v033.i07
King T, Lucchini S, Hinton JCD, Gobius K (2010) Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses. Appl Environ Microbiol 76:6514. https://doi.org/10.1128/AEM.02392-09
Kusnetsov J, Neuvonen L-K, Korpio T, Uldum SA, Mentula S, Putus T, Tran Minh NN, Martimo K-P (2010) Two Legionnaires’ disease cases associated with industrial waste water treatment plants: a case report. BMC Infect Dis 10:343. https://doi.org/10.1186/1471-2334-10-343
Lau HY, Ashbolt NJ (2009) The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol 107:368–378. https://doi.org/10.1111/j.1365-2672.2009.04208.x
Lund V, Fonahn W, Pettersen JE, Caugant DA, Ask E, Nysaeter A (2014) Detection of Legionella by cultivation and quantitative real-time polymerase chain reaction in biological waste water treatment plants in Norway. J Water Health 12:543–554. https://doi.org/10.2166/wh.2014.063
Ma J, Wang Z, Zang L, Huang J, Wu Z (2015) Occurrence and fate of potential pathogenic bacteria as revealed by pyrosequencing in a full-scale membrane bioreactor treating restaurant wastewater. RSC Adv 5:24469–24478. https://doi.org/10.1039/C4RA10220G
Maisa A, Brockmann A, Renken F, Lück C, Pleischl S, Exner M, Daniels-Haardt I, Jurke A (2015) Epidemiological investigation and case–control study: a Legionnaires’ disease outbreak associated with cooling towers in Warstein, Germany, August–September 2013. Eurosurveillance 20:30064. https://doi.org/10.2807/1560-7917.ES.2015.20.46.30064
Manz W, Amann R, Szewzyk R, Szewzyk U, Stenström TA, Hutzler P, Schleifer KH (1995) In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology 141(Pt 1):29–39. https://doi.org/10.1099/00221287-141-1-29
Medema G, Wullings B, Roeleveld P, van der Kooij D (2004) Risk assessment of Legionella and enteric pathogens in sewage treatment works. Water Sci Technol Water Supply 4:125–132
Nguyen TMN, Ilef D, Jarraud S, Rouil L, Campese C, Che D, Haeghebaert S, Ganiayre F, Marcel F, Etienne J, Desenclos J-C (2006) A community-wide outbreak of legionnaires disease linked to industrial cooling towers—How far can contaminated aerosols spread? J Infect Dis 193:102–111. https://doi.org/10.1086/498575
Nogueira R, Melo LF, Purkhold U, Wuertz S, Wagner M (2002) Nitrifying and heterotrophic population dynamics in biofilm reactors: effects of hydraulic retention time and the presence of organic carbon. Water Res 36:469–481
Nogueira R, Utecht K-U, Exner M, Verstraete W, Rosenwinkel K-H (2016) Strategies for the reduction of Legionella in biological treatment systems. Water Sci Technol 74:816–823. https://doi.org/10.2166/wst.2016.258
Ohno A, Kato N, Yamada K, Yamaguchi K (2003) Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl Environ Microbiol 69:2540–2547. https://doi.org/10.1128/AEM.69.5.2540-2547.2003
Ricke S (2003) Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult Sci 82:632–639. https://doi.org/10.1093/ps/82.4.632
Rowbotham TJ (1986) Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 22:678–689
Scheikl U, Sommer R, Kirschner A, Rameder A, Schrammel B, Zweimüller I, Wesner W, Hinker M, Walochnik J (2014) Free-living amoebae (FLA) co-occurring with legionellae in industrial waters. Eur J Protistol 50:422–429. https://doi.org/10.1016/j.ejop.2014.04.002
Sharaby Y, Rodríguez-Martínez S, Oks O, Pecellin M, Mizrahi H, Peretz A, Brettar I, Höfle MG, Halpern M (2017) Temperature-dependent growth modeling of environmental and clinical Legionella pneumophila multilocus variable-number tandem-repeat analysis (MLVA) genotypes. Appl Environ Microbiol. https://doi.org/10.1128/AEM.03295-16
Taylor M, Ross K, Bentham R (2009) Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb Ecol 58:538–547. https://doi.org/10.1007/s00248-009-9514-z
Taylor M, Ross K, Bentham R (2013) Spatial arrangement of legionella colonies in intact biofilms from a model cooling water system. Microbiol Insights 6:49–57. https://doi.org/10.4137/MBI.S12196
US EPA (2016) Microbial contaminants—contaminant candidate list and regulatory determination 4 [WWW document]. https://www.epa.gov/ccl/microbial-contaminants-ccl-4. Accessed 5 Sept 2018
US-EPA (2016) Technologies for legionella control in premise plumbing systems: scientific literature review. US-EPA, Washington, D.C.
Van Heijnsbergen E, Schalk JAC, Euser SM, Brandsema PS, Den Boer JW, De Roda Husman AM (2015) Confirmed and potential sources of Legionella reviewed. Environ Sci Technol 49:4797–4815. https://doi.org/10.1021/acs.est.5b00142
Wadowsky RM, Wolford R, McNamara AM, Yee RB (1985) Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water. Appl Environ Microbiol 49:1197–1205
Warren WJ, Miller RD (1979) Growth of Legionnaires disease bacterium (Legionella pneumophila) in chemically defined medium. J Clin Microbiol 10:50–55
Whiley H, Bentham R (2011) Legionella longbeachae and legionellosis. Emerg Infect Dis 17:579–583. https://doi.org/10.3201/eid1704.100446
WHO (2018) W.H.O. Legionellosis [WWW document]. http://www.who.int/news-room/fact-sheets/detail/legionellosis. Accessed 9 Oct 2018
Young KD (2006) The selective value of bacterial shape. Microbiol Mol Biol Rev 70:660–703. https://doi.org/10.1128/MMBR.00001-06
Zwietering MH, Jongenburger I, Rombouts FM, van’t Riet K (1990) Modeling of the bacterial growth curve. Appl Environ Microbiol 56:1875–1881
Acknowledgements
We thank Mrs. Karen Kock and Dr. Corinna Lorey from the Institute for Sanitary Engineering and Waste Management, Leibniz University Hannover, for performing the qPCR and FISH analyses, respectively. We are grateful to Mrs. Claudia Helle for her support during the growth experiments. This work was conducted with financial support from the industrial sector (Grant No.: CA-60451429).
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Caicedo, C., Verstraete, W., Rosenwinkel, KH. et al. Growth kinetics of environmental Legionella pneumophila isolated from industrial wastewater. Int. J. Environ. Sci. Technol. 17, 625–632 (2020). https://doi.org/10.1007/s13762-019-02482-5
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DOI: https://doi.org/10.1007/s13762-019-02482-5