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WP2: Active Months - February 2013 to January 2016

Reliable assays for the detection and characterization of viruses were developed. Procedures were established to estimate the inactivation of non-cultivable viruses. Typing methodologies were developed to characterize waterborne outbreaks.

WP 3: Active Months - February 2013 to January 2016

Molecular tools were developed for the identification and quantification of the major waterborne bacterial pathogens and successfully tested with water samples representing the drinking water supply chain.

Deliverable D3.5 
Analytical modules for targeted pathogens ready for integration into platforms:  Submission date: January 30, 2016
Authors: Manfred Höfle, HZI, Germany; Carla Pruzzo, UNIGE, Italy; Maria Jose Figueras, URV, Spain; *Antonio Martinez-Murcia, GPSTM, Spain; Jörg Peplies, Ribocon, Germany. 
*Lead beneficiary: Genetic PCR SolutionsTM, Alicante, Spain.


Deliverable 3.5 - Analytical modules for targeted pathogens ready for integration into platforms

1. Executive Summary 

The overall aim of WP 3 was to develop analytical modules for the identification and quantification the targeted bacterial pathogens which can be adapted in the platforms to enable commercialisation. These modules consist of sets of Standard Operational Protocols (SOPs) for the targeted pathogens to identify and quantify them at the genus, species or strain level. At a higher Technology Readiness Level (TRL) molecular test kits, containing all chemicals, enzymes and molecular standards for these tasks, were developed and validated in the relevant environments. 

Four different types of molecular markers/targets were explored for the identification and quantification of bacteria to the genus until the strain level: i) 16S ribosomal RNA genes, ii) highly conserved but variable functional genes (gyrB and rpoB), iii) specific virulence genes and iv) tandem repeats. The applicability of these four types of markers for molecular quantification of bacterial pathogens was assessed at three levels of validation: i) in-silico meaning comparison with International sequence data bases, ii) in-vitro meaning validation in the laboratory using reference strains, and iii) in-situ meaning validation with real water samples. The 16S rRNA gene, currently the gold standard for bacterial taxonomy, was successfully applied to identify the targeted bacteria at the genus and species level.  In combination with Next Generation Sequencing (NGS) technologies, genus-specific identification and quantification was demonstrated for all relevant species of the genus Pseudomonas, including the targeted species P. aeruginosa. In addition, pan-bacterial primers targeting the 16S RNA gene were applied in combination with NGS technologies to obtain a complete overview about all bacteria present in a given water sample to achieve the “detection of the unknown”. For the functional genes, the gyrase B (gyrB) gene was most promising because it could be demonstrated in-silico and in-vitro that it is possible to differentiate the major serovars of Salmonella enterica and identify clearly Escherichia coli and Shigella flexneri. In-situ application in a set of contaminated water samples confirmed this high resolution if specific primers were applied in a patho-printing approach using Illumina technology. New primers for the high resolution identification of Arcobacter butzleri and Campylobacter jejuni targeting the RNA polymerase B (rpoB) gene were developed and validated in-vitro using reference strains. Virulence genes for all targeted pathogenic species were assessed and validated for quantification by qPCR. A novel phylogenetic marker for a virulence related gene was discovered for Vibrio cholerae based on which a highly specific qPCR assay was developed and validated. The value of tandem repeats was explored in a MLVA (Multiple-Locus Variable number tandem repeat (VNTR) Analysis) approach for Pseudomonas aeruginosa and Legionella pneumophila because of their commercial relevance. Both MLVA assays could be validated in-vitro with a comprehensive set of reference strains using multiplex PCR with fluorescent primers and capillary electrophoresis (CE) for detection and separation of the amplicons. 

Molecular quantification kits and genome standards for all targeted species were validated and are now commercially available from SMEs. In conclusion, this deliverable report demonstrates that WP 3 achieved the final Milestone MS 26 “Molecular tools for high resolution identification” of bacterial targeted species & genus-specific bacterial fingerprints available for integration into platforms developed in Cluster 2 and for field trails in Cluster 3. 

Implications of the results of Deliverable Report D3.5

Implications of the results for the Work Package (WP 3) 
This deliverable report provides a large variety of Standard Operational Protocols (SOPs) to identify and quantify the targeted bacterial pathogens at the genus, species or strain level. Molecular test kits, containing all chemicals, enzymes and molecular standards for these tasks, were developed and validated. In essence, it is the final report of the major results of WP3.

Implications of the results for the Cluster 1, Molecular detection of bacterial pathogens 

Overall, D3.5 comprises the results of work at a higher Technology Readiness Level (TRL) that fulfil the aim of WP 3 within Cluster 1, related to the bacteria selected in the project. This deliverable report delivers a set of analytical modules developed for the identification and quantification of the targeted bacterial pathogens which can be adapted in the platforms developed of Cluster 2 to enable commercialisation.

Implications of the results for the whole project 

This deliverable report provides the analytical modules developed in WP3 at two different Technology Readiness Levels (TRL): i) molecular identification and quantification technologies validated in relevant environments and SOPs developed, and ii) commercially available molecular test kits and molecular standards validated in relevant environment.  In conclusion, this deliverable report demonstrates that WP 3 achieved the final Milestone MS 26 “Molecular tools for high resolution identification” of bacterial targeted species and genus-specific bacterial fingerprints available for integration into platforms developed in Cluster 2 and for field trails in Cluster 3.

Indicate key external stakeholders interested in the results of Deliverable Report 3.5 
SOPs are of interest to partners of Cluster 2 and 3, and consequently to external stakeholders (large and small water suppliers, food industry, and regulators). Molecular quantification kits and genome standards for all targeted bacterial species were validated and are now commercially available from SMEs which should be specifically advertised by the coordinator.

Which internal partners should your deliverable be sent to?   
All the 39 internal partners in Aquavalens should receive a copy, it will be particularly important to those active in Clusters 2 and 3. This deliverable report will help with planning the integration of molecular assays for the detection of bacteria into the platforms developed in Cluster 2 and help perform the field trials in Cluster 3.     



International Publications of WP 3 until 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

WP 4: Active Months - February 2013 to January 2016

This work package has validated and applied molecular targets for detection and human infectivity potential of the protozoan parasites; Cryptosporidium, Giardia and Toxoplasma, and investigated targets that might be associated with virulence. 

Deliverable D4.4 (Version 2, 02/12/15)

Molecular tools for parasite lineage to support source tracking in outbreak investigations: Authors: Rachel Chalmers, Sophie May, Guy Robinson, Gregorio Perez, Stephen Hadfield (PHL), Frank Katzer, Alison Burrells, Beth Wells, Emily Hotchkiss, Elisabeth Innes (MRI), Kevin Tyler, Johanna Nader, Maha Bouzid (UEA).

D4.4 Executive Summary

Waterborne outbreaks of human disease have occurred caused by the protozoan parasites Cryptosporidium species, Giardia duodenalis and Toxoplasma gondii in drinking water.  To investigate sources of contamination, transmission routes and outbreaks of disease, high resolution genotyping is needed. To underpin the development of standardised, multi-locus schemes, this work focussed mainly on the major human pathogens Cryptosporidium hominis and Cryptosporidium parvum, especially the zoonotic C. parvum. The initial focus was on multiple-locus variable-numbers of tandem repeats analysis (MLVA). Potentially-useful loci were identified and examined in silico for suitability for inclusion in inter-laboratory MLVA schemes. An economical capillary electrophoresis (CE) platform (QIAxcel, Qiagen) was optimised for performance with single round PCR amplicons, and epidemiological utility was demonstrated by the investigation of a panel of sporadic and outbreak-related clinical samples. The loci were tested in vitro in different laboratories using different CE machines through the distribution of diverse C. parvum DNA. Although many loci were more complex than previously thought, the use of sequenced reference standards enabled consistent allele calling based on adjusted fragment sizes. Additionally, the Cryptosporidium whole genomes generated in Aquavalens D4.1 were investigated for new subtyping markers, and suitable candidates were identified for potential inclusion in MLVA.

For T. gondii, we developed and tested a multiplex-nested-PCR-RFLP assay for genotyping based on five markers that originate from four distinct gene loci within the parasite genome. Although sensitive, being able to detect single parasites within a PCR reaction, the assay was less sensitive than the 529 bp qPCR used for T. gondii enumeration. The assay did not provide enough discriminatory power to distinguish between Type II strains of the parasite which are the most commonly found strains within Europe.

WP 5: Active Months - May 2013 to January 2016

A data matrix based on the first sampling results has been created. This was used to develop MST predictive models and select MST markers for further testing in 2015.

Deliverable D5.4

MST predictive models validated at European level.

Authors: Leena Maunula1, Anicet R. Blanch2, Lluis A. Belanche3,

1 University of Helsinki, Finland, 2 University of Barcelona, Spain

3  Polytechnic University of Catalonia, Spain


Abbreviations:  Mitochondrial DNA markers: Human-specific (HMMit), Porcine (PGMit), Bacteroidetes DNA: Human-specific (HF183Taqman), Ruminant (BacR), Porcine (Pig2Bac), Bifidobacterium DNA: Human-specific (HMBif), Bovine (CWBif), Poultry (PLBif), Total (TLBif), Norovirus RNA (NoV)

Results and Discussion

The selected predictive models were evaluated for prediction and their corresponding accuracy was estimated by repeated rounds of k-CV, as explained above. Furthermore, four validated MST predictive models at the five participant countries (Austria, Germany, Finland, Portugal and Spain) were selected based on molecular markers only, in order to be considered for future use on technological platforms.

These selected models determine a subset of nine molecular parameters (see below and in abbreviations) to be considered as preliminary candidates for the final list of molecular targets on MST due to the end of the WP5 (Month 32). However, the final list of selected MST markers to include in the technological platforms will be confirmed through the second sampling campaign.

The validated MST molecular-based models (linear or non-linear) and the corresponding scenarios providing solutions are:

Linear Discriminating Analysis

Human vs Non-human  /  point source

HMBif, PLBif, TLBif, Pig2Bac, HF183TaqMan  (accuracy 100% by LOOCV)

Four sources  /  point source

HMBif, BacR, Pig2Bac  (accuracy 100% by LOOCV)

Non-linear Analysis (Random Forest)

Human vs Non-human

NoV, PGMit (accuracy 88.5% by 10x10 CV)

Four sources

NoV, PGMit, CWBif, HMBif, TLBif/CWBif (accuracy by 79.1% by 10x10 CV)

Although adding some additional molecular markers to the subsets selected could increase accuracy, it could not be worthy when considering costs. If other combinations of molecular markers are requested with the purpose of implementation on technological platforms, it could be possible to select other predictive models out of the already performed calculations at close accuracy values.