Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca, a drinking water source in the north-eastern part of Turkey

  • Reyhan Akçaalan
  • Meric Albay
  • Latife Koker
  • Julia Baudart
  • Delphine Guillebault
  • Sabine Fischer
  • Wilfried Weigel
  • Linda K. MedlinEmail author


Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project μAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.


Phylochips Microarray Freshwater pathogens Cyanobacteria Molecular barcodes 


Funding information

This work was supported by the EU μAQUA project (FP7-KBBE-2010-4, 265409).

Supplementary material

10661_2017_6314_MOESM1_ESM.doc (411 kb)
ESM 1 (DOC 411 kb)
10661_2017_6314_MOESM2_ESM.pdf (367 kb)
ESM 2 (PDF 367 kb)


  1. Akçaalan, R., Köker, L., GürevIn, C., & Albay, M. (2014). Planktothrix rubescens, a perennial presence and toxicity in Lake Sapanca. Turkish Journal of Botany, 38, 782–789.CrossRefGoogle Scholar
  2. Allgaier, M., & Grossart, H. P. (2006). Diversity and seasonal dynamics of Actinobacteria populations in four lakes in northeastern Germany. Applied and Environmental Microbiology, 72, 3489–3497.CrossRefGoogle Scholar
  3. Altüg, G., Yardimci, C. H., Okgerman, H., & Tarkan, S. A. (2006). Levels of bacterial metabolic activity, indicator (Coliform, Escherichia coli) and pathogen bacteria (Salmonella spp.) in the surface water of Sapanca Lake, Turkey. Journal of the Black Sea/Mediterranean Environment, 12, 67–77.Google Scholar
  4. Aw, T. G., & Rose, J. B. (2012). Detection of pathogens in water, from phylochips to qPCR to pyrosequencing. Current Opinion in Biotechnology, 23, 422–430.CrossRefGoogle Scholar
  5. Baker-Austin, C., Trinanes, J., Gonzalez-Escalona, N., & Martinez-Urtaza, J. (2017). Non-cholera Vibrios, the microbial barometer of climate change. Trends in Microbiology, 25, 76–84.CrossRefGoogle Scholar
  6. Baudart, J., Guillebault, D., Meilke, E., Meyer, T., Tandon, N., Fischer, S., Weigel, W., & Medlin, L. K. (2016). Microarray (phylochip) analysis of freshwater pathogens at several sites along the Northern German coast transecting both estuarine and freshwaters. Applied Microbiology and Biotechnology, 101, 871–886.CrossRefGoogle Scholar
  7. Bradford, S. A., & Schijven, J. (2002). Release of Cryptosporidium and Giardia from dairy calf manure, impact of solution salinity. Environmental Science and Technology, 36, 3916–3923.CrossRefGoogle Scholar
  8. Caraux, G., & Pinloche, S. (2005). PermutMatrix, a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics, 21, 1280–1281.CrossRefGoogle Scholar
  9. Certad, G., Dupouy-Camet, J., Gantois, N., Hammouma-Ghelboun, O., Pottier, M., Guyot, K., Benamrouz, S., Osman, M., Delaire, B., Creusy, C., Viscogliosi, E., Dei-Cas, E., Aliouat-Denis, C. M. & Follet, J. (2015). Identification of Cryptosporidium species in fish from Lake Geneva (Lac Léman) in France. PLoS One, 10.
  10. Cai, T., Jiang, L., Yang, C., & Huang, K. (2006). Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China. FEMS Immunology & Medical Microbiology, 46, 180–186.CrossRefGoogle Scholar
  11. Chomczynski, P., & Sacchi, N. (1987). The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction twenty-something years on. Nature Protocols, 1, 581–585.CrossRefGoogle Scholar
  12. Clifford, R. J., Milillo, M., Prestwood, J., Quintero, R., Zurawski, D. V., Kwak, Y. I., Waterman, P. E., Lesho, E. P., & Mc Gann, P. (2012). Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR. PLoS One, 7, e48558.CrossRefGoogle Scholar
  13. Davis, P. A., Dent, M., Parker, J., Reynolds, C. S., & Walsby, A. E. (2003). The annual cycle of growth rate and biomass change in Planktothrix spp. in Blelham Tarn., English Lake District. Freshwater Biology, 48, 852–867.CrossRefGoogle Scholar
  14. Da-Silva, E., Barthelmebs, L., & Baudart, J. (2016). Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA. Environmental Science and Pollution Research, 24, 5690–5700.CrossRefGoogle Scholar
  15. Dittami, S. M., & Edvardsen, B. (2013). GPR-analyzer, a simple tool for quantitative analysis of hierarchical multispecies microarrays. Environmental Science and Pollution Research, 20, 6808–6815.CrossRefGoogle Scholar
  16. EPA. (2014). Cyanobacteria and cyanotoxins, information for drinking water systems. EPA 810F11001. Available online at
  17. Frahm, E., & Obst, U. (2003). Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples. Journal of Microbiological Methods, 52, 123–131.CrossRefGoogle Scholar
  18. Gescher, G., Metfies, K., & MedlIn, L. K. (2008). The ALEX Chip—development of a DNA chip for identification. Harmful Algae, 7, 485–494.CrossRefGoogle Scholar
  19. Greer, B., McNamee, S. E., Boots, B., Cimarelli, L., Guillebault, D., Helmi, K., Marcheggiani, S., Panaiotov, S., Breitenbach, U., Akcaalan, R., Medlin, L. K., Kittler, K., Elliot, C. T., & Campbell, K. (2016). A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems. Harmful Algae, 55, 31–40.CrossRefGoogle Scholar
  20. Griffitt, K. J., Noriea, N. F., Johnson, C. N., & Grimes, D. J. (2011). Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization. Journal of Microbiological Methods, 85, 114–118.CrossRefGoogle Scholar
  21. Hill, V. R., Polaczyk, A. L., Hahn, D., Narayanan, J., Cromeans, T. L., Roberts, J. M., & Amburgey, J. E. (2005). Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants. Applied and Environmental Microbiology, 71, 6878–6884.CrossRefGoogle Scholar
  22. Karlson, B., Cusack, C., & Bresnan, E. (Eds). (2010). Microscopic and molecular methods for quantitative phytoplankton analysis. IOC Manuals and Guides, no. 55 (110 pages). Paris: Intergovernmental Oceanographic Commission of ©UNESCO.Google Scholar
  23. Kegel, J. U., Del Amo, Y., & Medlin, L. K. (2013). Introduction to project MIDTAL, its methods and samples from Arcachon Bay., France. Environmental Science and Pollution Research, 20, 6690–6704.CrossRefGoogle Scholar
  24. Kegel, J. U., Del Amo, Y., Costes L. & Medlin L. K. (2016). Monitoring toxic microalgae in Arcachon Bay in France by microarrays. Microarrays, Featured Papers 2, 1–23.Google Scholar
  25. Lawton, L. A., Edwards, C., & Codd, G. A. (1994). Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst, 119, 1525–1530.CrossRefGoogle Scholar
  26. Lewis, J., Medlin, L. K., & Raine, R. (2012). MIDTAL (microarrays for the detection of toxic algae). A protocol for a successful microarray hybridisation and analysis. Germany: Koeltz., Koenigstein.Google Scholar
  27. Kurmayer, R., Blom, J. F., Deng, L., & Pernthaler, J. (2015). Integrating phylogeny., geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix. The International Sosciety of Micorbial Ecology Journal, 9, 909–921.Google Scholar
  28. Lindahl, J. F. & Grace, D. (2015). The consequences of human actions on risks for infectious diseases, a review. Infection, Ecology, and Epidemiology, 5.
  29. Marcheggiani, S., D’Ugo, E., Puccinelli, C., Giuseppetti, R., D’Angelo, A. M., Gualerzi, C. O., Spurio, R., MedlIn, L. K., Guillebault, D., Baudart-Lenfant, J., Weigel, W., Helmi, K., & Mancini, L. (2015). Detection of emerging and re-emerging pathogens in surface waters close to an urban area. International Journal of Environmental Research and Public Health, 12, 5505–5527.CrossRefGoogle Scholar
  30. Medlin, L. K. (2016). Mini review: molecular techniques for identification and characterization of marine biodiversity. Annals of Marine Biology and Research, 3(2), 1015.Google Scholar
  31. Medlin, L. K. (2013). Note: steps taken to optimise probe specificity and signal intensity prior to field validation of the MIDTAL microarray for the detection of toxic algae. Environmental Science and Pollution Research.
  32. Medlin L. K., Guillebault, D., Mengs, G., Garbi, C., Dejana, L., Fajardo, C. & Martin, M. (2017). New molecular tools, application of the mAQUA phylochip and concomitant FISH probes to study freshwater pathogens from samples taken along the Tiber River., Italy. Proceedings of 17 th Water Quality Conference, 221: in press.Google Scholar
  33. McLoughlin, K. S. (2011). Microarrays for pathogen detection and analysis. Brief Functional Genomics, 10, 342–353.CrossRefGoogle Scholar
  34. Oliver, J. D. (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiological Review, 34, 415–425.CrossRefGoogle Scholar
  35. Oliveira, T. C. R. M., Barbut, S., & Griffiths, M. W. (2005). Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR. International Journal of Food Microbiology, 104, 105–111.CrossRefGoogle Scholar
  36. Oudra, B., Loudiki, M., Vasconcelos, V., Sabour, B., Sbiyyaa, B., Oufdou, K., & Mezrioui, N. (2002). Detection and quantification of microcystins from cyanobacteria strains isolated from reservoirs and ponds in Morocco. Environmental Toxicology, 17, 32–39.CrossRefGoogle Scholar
  37. Paniel, N., Baudart, J., Hayat, A., & Barthelmebs, L. (2013). Aptasensor and genosensor methods for detection of microbes in real world samples. Methods, 64, 229–240.Google Scholar
  38. Rodriguez, I., Fraga, M., Alfonso, A., Guillebault, D., Medlin L., Baudart, J., Jacob, P., Helmi, K., Meyer, T., Breitenbach, U., Holden, N. M., Boots, B., Spurio, R., Cimarelli, L., Mancini, L., Marcheggiani, S., Albay, M., Akcaalan, R., Köker, L., & Botana, L. M. (2016). Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods. Environmental Toxicology and Chemistry, 36(3), 645–654.
  39. Santhi, N., Pradeepa, C., Subashini, P., & Kalaiselvi, S. (2013). Automatic identification of algal communities from microscopic images. Bioinformatics and Biology Insights, 7, 327–334.CrossRefGoogle Scholar
  40. Sidhu, J. P., Hodgers, L., Ahmed, W., Chong, M. N., & Toze, S. (2012). Prevalence of human pathogens and indicators in stormwater runoff in Brisbane, Australia. Water Research, 46, 6652–6660.CrossRefGoogle Scholar
  41. Sivonen, K., & Jones, G. (1999). Cyanobacterial toxins. In I. Chorus & J. Bartram (Eds.), Toxic Cyanobacteria in Water, A Guide to their Public Health Consequences., Monitoring and Management (pp. 41–91). London: E & F Spon.Google Scholar
  42. Suda, S., Watanabe, M. M., Otsuka, S., Chong, M. N., & Toze, S. (2002). Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria. Intternational Journal of Systematic Evolution and Microbiology, 52, 1577–1595.Google Scholar
  43. Tokat, B. (2010). Temporal and spatial distribution of picophytoplankton in Sapanca lake. MSc Thesis. Istanbul University, Institute of Graduate Studies in Science and Engineering.Google Scholar
  44. Utermöhl, H. (1958). Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mittleilungen Verhandlungen des Internationalen Verein Limnologie, 9, 1–38.Google Scholar
  45. Van Apeldoorn, M. E., van Egmond, H. P., Speijers, G. J. A., & Bakker, G. J. I. (2007). Toxins of cyanobacteria. Molecular Nutritional Food Research, 51, 7–60.CrossRefGoogle Scholar
  46. Van der Gucht, K., Van de Kerckhove, T., Vloemans, N., CousIn, S., Muylaert, K., Sabbe, K., Gillis, M., Declerk, S., De Meester, L., & Vyverman, W. (2005). Characterization of bacterial communities in four freshwater lakes differing in nutrient load and food web structure. FEMS Microbiology Ecology, 53, 205–220.CrossRefGoogle Scholar
  47. Van Der Waal, D., Guillebault D., Alfonso D., Rodríguez, I., Botana, L. & Medlin L. K. (2017). μAqua microarrays for phylogenetic and toxin expression of cyanobacteria with validation by cell counts and UPLC/MS-MS. Harmful Algae, in press.Google Scholar
  48. WHO (2006). Guideline for drinking water quality, vol. 1, 3rd edn. Geneva: Recommendations Nonserial Publication, ISBN-13, 9789241546744.Google Scholar
  49. Zwart, G., Crump, B. C., Agterveld, M. P., Hagen, F., & Han, S.-K. (2002). Typical freshwater bacteria, an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquatic Microbial Ecology, 28, 141–155.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Reyhan Akçaalan
    • 1
  • Meric Albay
    • 1
  • Latife Koker
    • 1
  • Julia Baudart
    • 2
  • Delphine Guillebault
    • 3
  • Sabine Fischer
    • 4
  • Wilfried Weigel
    • 4
  • Linda K. Medlin
    • 5
    Email author
  1. 1.Faculty of Aquatic SciencesIstanbul UniversityIstanbulTurkey
  2. 2.Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Observatoire OcéanologiqueBanyuls/MerFrance
  3. 3.Microbia EnvironnementBanyuls sur MerFrance
  4. 4.Scienion AG VolmerstrBerlinGermany
  5. 5.Marine Biological Association of the UKPlymouthUK

Personalised recommendations