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Cluster 1 Summary

Cluster 1 includes WP 2, 3, 4 and 5. The primary objective in this cluster is to identify appropriate molecular targets or markers and gene sequences that could be used to provide insights into the presence of pathogens in water, their viability and virulence or could be used for strain discrimination in microbial typing and source tracking. Because the issues facing microbiologists working with viruses, bacteria and protozoa can be quite different we have chosen to have separate work packages covering each of these categories of microorganism. In addition we have an independent WP on source tracking as this could include all three categories and also non microbiological markers. We have restricted our research to pathogens acquired orally. Targeted pathogens include: Norovirus, Rotavirus, Adenovirus and Hepatitis A and E viruses (WP2), A. butzleri, C. coli, C. jejuni, V. cholerae, P. aeruginosa, S. enterica, pathogenic E. coli and indicator bacteria (WP3), and Cryptosporidium, Giardia and Toxoplasma (WP4).


WP2: Applied Virology - Molecular detection, quantification and typing of infectious waterborne viruses. Active Months: February 2013 to January 2016


  • To establish threshold maximum acceptance levels for genome copies of health-significant waterborne viruses that cannot be grown in cell cultures so that they represent an acceptable risk for the consumers.
  • To indirectly ascertain the infectivity of viruses detected by molecular procedures. Several sample pre-treatments prior to molecular detection will be evaluated in order to provide a better estimation of infectious virus numbers. Additionally systems to evaluate specific virus nucleic acid or protein damage caused by some virucidal treatments will be developed.
  • To determine the genetic diversity within the target viruses and how such diversity could be exploited in outbreak investigation to link human and environmental strains. Genetic characterization of virus isolates will determine the potential zoonotic origin of waterborne viruses.


Del2.1 Determine maximum acceptance levels for genome  copy numbers of viruses in water

Del2.2 Design of variation tolerant primers and probes based on the genomic variation of viruses

Del2.3 Provide a correct estimation of infectious viruses through determination of genome copy numbers

Del2.4 Establish molecular methods to evaluate the efficacy of virucidal treatments on viruses

Del2.5 Elucidate the epidemiology and transmission of enteric viruses through genotype determination. Currently Confidential.


WP3: Applied Bacteriology - Assessment of load and virulence of waterborne bacterial pathogens. Active Months: February 2013 to January 2016


  • To develop and apply advanced molecular tools for high-resolution identification and quantification of waterborne bacterial pathogens.
  • To develop and apply molecular markers for the identification of single strains of all major species of waterborne bacterial pathogens.
  • To assess viability of pathogenic bacteria in freshwater by molecular and classical methods.
  • To assess virulence and infectivity of pathogenic bacteria in freshwater using molecular methods and experimental in-vitro assays.


Del3.1 Standard monitoring methods for identification of the targeted bacteria harmonized & SOPs developed

Del3.2 Genus-specific pyroprinting developed and validated

Del3.3 Advanced molecular tools for high resolution identification and in-situ application developed

Del3.4 Virulence maker expression and infectivity assessed for the targeted bacterial species

Del3.5 Analytical modules for targeted pathogens ready for integration into platforms


International Publications of WP 3 up to January 31, 2016.

Fisher, J.C., Levican, A., Figueras, M.J., McLellan., S.L. 2014. Population dynamics and ecology of Arcobacter in sewage. Frontiers in Microbiology 5: 525; doi:10.3389/fmicb.2014.00525
Figueras, M.J., Beaz-Hidalgo, R., Hossain, M.J., Liles, M.R. 2014.Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis. Genome Announcements 2: e00927-14; doi:10.1128/genomeA.00927-14 
Figueras, M.J., Levican, A., Pujol, I., Ballester, F., Rabada, Quilez, M.Jm, Gomez-Bertomeu, F. 2014. A severe case of persistent diarrhoea associated with Arcobacter cryaerophilus but attributed to Campylobacter sp. and a review of the clinicalinc idence of Arcobacter spp.. New Microbes New Infect. 2:31-37; doi: 10.1002/2052-2975.35. 
Hossain, M.J., Beaz-Hidalgo, R., Figueras, M.J., Liles, M.R. 2014. Draft genome sequences of two novel Aeromonas species recovered in association with cyanobacterial blooms.  Genome Announcements 2: e01181-14; doi: 10.1128/genomeA.01181-14
Vezzulli L., E. Pezzati, I. Brettar, M. G. Höfle, Pruzzo, C. 2015. Effects of global warming on Vibrio ecology. Microbiol. Spectrum 3: VE-0004-2014 doi:10.1128/microbiolspec.VE-0004-2014
Beaz-Hidalgo, R., Hossain, M.J., Liles, M.R., Figueras, M. J. 2015. Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for Aeromonas genomes in the GenBank database.  PLoS ONE 10:e0115813; doi:10.1371/journal.pone.0115813
Levican A, Rubio-Arcos S, Martinez-Murcia A, Collado L, Figueras M. J. 2015. Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment. Systematic and Applied Microbiology    38:30-35; doi:10.1016/j.syapm.2014.10.011.
Martinez-Murcia, A., Lamy, B. 2015. Molecular Diagnostics by Genetic Methods.     Aeromonas, Caister Academic Press, Chapter 8, p. 180-195, ISBN: 978-1-908230-56-0
Vezzulli L, Stauder M, Grande C, Pezzati E, Verheye HM, Owens NJ, Pruzzo C. 2015. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, non-O1/non-O139 in environmental, stool, and historical continuous plankton recorder samples. PLoS ONE 10: e0123983; doi:10.1371/journal.pone.0123983
Lesnik R., Brettar I., Höfle MG. 2015. Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily. ISME Journal; DOI: 10.1038/ismej.2015.199    
Martínez-Murcia A., Beaz-Hidalgo R., Navarro A., Carvalho M.J., Aravena-Román M., Correia A., Figueras M.J., Saavedra M. J. 2016. Aeromonas lusitana sp. nov., isolated from untreated waters and vegetables. Current Microbiology, 72:795–803  


WP4: Applied parasitology – Detection, virulence assessment and sub-typing of major waterborne protozoa. Active Months: February 2013 to January 2016


  • To identify and develop effective molecular targets for detection, virulence and infectivity assessment and high resolution genotyping of major waterborne protozoa.
  • To identify for Cryptosporidium spp. genetic loci displaying variation correlating with virulence and host specificity and to validate markers by correlation of expression with pathogenicity and virulence in symptomatic animal hosts.
  • To identify putative virulence factors for Giardia spp. by comparative evaluation of strain specific secretomesand to validate those with ex-vivo models of intestinal epithelia for assay development.
  • To develop molecular tools to genotype, quantify and determine the infectivity and viability of Toxoplasma spp.oocysts in water generated from a mouse to cat infection model.


Del4.1 New genome sequences for Cryptosporidium

Del4.2 Genetic definition of experimentally validated giardia secretome

Del4.3 Molecular tools for parasite quantity and species, Giardia virulence and T. gondii infectivity

Del4.4 Molecular tools for parasite lineage to support source tracking in outbreak investigations




WP5: Microbial Source Tracking - Combining cultivation-dependent and molecular techniques. Active Months: April 2013 to January 2016


  • To evaluate the feasibility of novel MST indicators and particularly those using molecular methodologies. 
  • To apply inductive learning and statistical methods to select and define the best indicators and their combinations for modelling and distinguishing faecal pollution from more than two origins.
  • To develop MST predictions models and verify their applicability at European level.
  • To evaluate the inclusion of the selected MST indicators in the developed platforms (cluster 2).


Del5.1  Booklet of methodologies and protocols for internal use of participants.

Del5.2  Data set on the distribution of different chemical and microbial parameters in surface waters.

Del5.3  List of the best subsets of indicators. Confidential.

Del5.4  MST predictive models validated at European level. Confidential.

Del 5.5 List of selected MST indicators or markers to include in the technological platforms. Confidential.