Detection – Assessment and

development of a portfolio of

pathogen detection methods

SLU, UB, GPS, DTU, IPU, TZW, microLAN, Vermicon, MUW

From the call text

“The new approaches should be based on molecular methods and complement the current time-

consuming microbiological techniques”

“Highly standardised methods are essential, validated with certified molecular reference material.”

“The approaches will need to address the issue of inhibition of molecular methods and assess the

significance of any positive detection.”

”The combination of molecular techniques with electronic sensors will also be investigated.”

“The new techniques will result in detailed insight into the pathogen load, the hygienic quality and the

specific microbial strains (viruses, bacteria, protozoa) responsible for outbreaks of waterborne infections.”

” They will lead to better understanding of the sources, infectivity and virulence of these strains.”

“The efficacy of the new techniques has to be demonstrated.”

From the application

Objective 4: to develop a portfolio of detection methods on a

range of different platforms suitable for various applications

(source water, treated water for drinking and food production

surface, ground, tap, processing water), laboratories (high, low

tech) and water management systems (on-site, online

measurement, large, small distribution systems).

1, Tillämpad mikrobiologi: identifiering av molekylära

mål, markörer och gensekvenser som ger bättre insyn i

förekomsten av patogener i vatten, deras viabilitet och

virulens.

2, Plattformar för detektion: utveckling för att påvisa

dessa målsekvenser i vatten, automatisering,

integrering och standardisering av metoderna så att de

kan användas i kontrollprogram.

3, Provtagning: utformning av provtagningsprogram för

stora och små vattenverk samt vatten för livsmedels-

produktion.

4, Effekt, syntetisering av resultat: öka kunskaperna

med hälsorisker från vatten, hur dessa påverkas av nya

patogener och klimatförändring. Vilken effekt har

inrättandet av vattensäkerhetsplaner och hur kan nya

metoder förbättra och förenkla inrättandet av dem?

Time

Pre

cis

ion

Pollution

Faecal

pollution

Pathogen

species

Pathogen

virulence

Typing

min hours days weeks

Outbreak

typing

ATP-biosensor

ICM-biosensor

BACTcontrol

VOCMA

FISH/SPC

in situ PCR-FISH(RT)-qPCR

XX

X

Developed portfolio of detection methods

On the way to on-line monitoring….BACTcontrol and ATP biosensor• In-line

• Real-time - fast respons vs. incubation based methods

• Continuous - High frequency data – vs. grab samples

• On-line - communication – remote monitoring

– Sensors or sensor-arrays combined with

Information-Communication-Technologies (ICT)

• Documentation of water quality

• Process control

• Trend monitoring

• Early warning

Based on Patented Fluorescent measurement of specific enzymatic activity:

1. ß-Glucuronidase-> indicates E.coli activity

2. ß-Galactosidase-> indicates Coliform activity

3. Alkaline Phosphatase -> indicates Total Activity

4. ß-Glucosidase -> indicates Enterococci

Example: monitoring in River Rhine

Campylobacter coli

Fluorescence in situ Hybridisation (FISH)

Improved automatic scanning of filters

Campylobacter coli

Fluorescence in situ Hybridisation (FISH)

Specific probes are available for:

• Thermophilic Campylobacter species

• E. coli

• Coliforms

• Pseudomonas aeruginosa

• Total viable cells

Conclusions and outcomes:

Standardised molecular pathogen detection- real-time PCR

Prioritised pathogens

Lab on a chip for

PoC analysis

Wider impact on water safety of improved techniques to detect

waterborne pathogens, in large water supplies

Robert Pitchers, Clàudia Puigdomènech, Janis Eglitis, Gemma Saucedo, Beate Hambsch

Aquavalens: Management of drinking water quality

WP10-Large Systems

Aquavalens WP 10: Objectives

Implement and test the potential use of developed platforms and analytical technologies, developed previously, for the monitoring of real water samples from large drinking water systems. The specific objectives are to:

1. Identify a suitable range of water supply systems from across Europe to provide a representative range of conditions.

2. Develop procedures for platform integration on these water supplies.

3. Obtain data on the occurrence of waterborne pathogens in these types of source waters.

4. Establish methods and tools for risk management in large water systems.

WP10-Large Systems

Online systems

Selection of techniques

– Primary concentration: RexeedTM -25 A

� Commercially available filter

� Large volume filtration (up to 1000 L)

� Virus, bacteria and protozoa

- BACTcontrol (MicroLAN)

� Results available in 2 hours -> Early

Warning System

� Specific (E.coli/coliform bacteria) and non-

specific bacterial activity (total activity)

Recovery and concentration techniques

– PCR (Polymerase Chain Reaction)

� Molecular method

� Measures genetic material (live and dead

microorganisms)

– FISH (Fluorescence in situ hybridisation)

� Microscopy technique combined with

molecular method

� Detects single cells

Detection techniques

– Nucleic acid extraction: NucliSENS® and Qiagen

� Total nucleic acid extraction (DNA and RNA)

– Secondary concentration:

• Centricon® 70 plus

� Large volume processing (batches of 70

mL)

� Three kingdoms and drinking water

• VivaSpin® 15R

� Previous good experience from other

AQV partners

� Suitable for all three kingdoms

• PEG precipitation

� Good recovery in surface waters

� Suitable for all three kingdomsPCR

10-1000L

Eluate

600 mL

2nd conc.

Conc.

DNA extr.

FISH

Verif. methods

WP10-Large Systems

Demonstration sites:

Drinking Water Treatment Plants

UK siteGerman site Danish site

Spanish site

Raw water Raw water

Raw water

Post DAF

Treated WaterGAC filter

Sand filter

Sand

filter

Treated Water

After Sedimentation

Treated Water

Raw water

Treated

water

WP10-Large Systems

Aquavalens WP 10: Experimental plan; Investigated parameters and pathogens

o Viruses

� Ceeram kits: Norovirus GI, GII and

Hepatitis A virus

o Bacteria

• GPS kits: E. coli, Campylobacter spp,

C. jejuni, P. aeruginosa, Salmonella

spp, L. pneumophila

o Protozoa

� Ceeram kits: Giardia spp,

Cryptosporidium spp

� GPS kits: G. intestinalis,

Cryptosporidium spp, T. gondii

� Total cell count (DAPI staining)

� Viable cell count (FISH)

� E. coli (FISH)

� thermophilic Campylobacter (FISH)

PCR FISH and microscopy techniques

Verification analyses

� E. coli

� Coliform bacteria

� Campylobacter spp

� C. perfringens

� Somatic coliphages

� Enterococci

� P.aeruginosa

� L.pneumophila

� Giardia spp

� Cryptosporidium spp

WP10-Large Systems

Outline of results

WP10-Large Systems

PCR – bacteria – E.coli

WP10-Large Systems

PCR - noroviruses

Spanish site: Norovirus GI results

WP10-Large Systems

Fluorescence in-situ hybridisation (FISH)

WP10-Large Systems

BACTcontrol – total coliforms module

German site: Comparison of BACTcontrol total coliform activity and total coliform numbers

WP10-Large Systems

BACTcontrol – Total Activity (TA) module

Spanish site: Comparison of BACTcontrol TA measurements in all water matrices tested

0-10 pmol/min

Typical values of

chlorinated water

Treated

water

Sensor platform

(Network)GAC filterOzonated

water

Sand filter

Ozonation

effect

GAC

regrowth

Chlorination +

membranes

effect

WP10-Large Systems

BACTcontrol – Total Activity (TA) module

Spanish site:

Quality events detection of

BACTcontrol through TA

measurements in sand filters

Observations:

• Relationship between TA and

ammonium peaks

• Bacterial activity rise

detected after raw water pre-

treatment

Heavy rainfall episode : Wastewater discharges into the river

*First steps of the DWTP

(afterwards, advanced

treatment takes place)

WP10-Large Systems

Lessons learnt? Where, when and how to use the new

techniques

WP10-Large Systems

1st concentration Conclusions

1st CONCENTRATION STEP: REXEED FILTRATION

• RexeedTM filtration has proved to be a good tool in terms of recovery

and concentration for viruses and bacteria in process and drinking

water with large water volumes.

• Concentrating with RexeedTM is not useful for very loaded water

sources for bacteria (i.e Spanish site raw water). In addition. Humic

substances in water sources may cause excessive foam formation

during elution.

• Concentrating with RexeedTM has enabled verification analyses,

such as Colilert®, to be more sensitive, thus lowering the detection

limits significantly.

• RexeedTM did not present any clogging problems in any of the

sampling campaign, even when filtering 1000 L (drinking water)

Lessons learnt? Where, when and how to use

the new techniques

WP10-Large Systems

Results:

Recovery and concentration techniques

Primary concentration: RexeedTM filtration Secondary concentration• Centricon®Plus- 70: Processes 15-20 times higher

volumes (140-210 mL instead of usual 10 mL) than

conventional centrifugation techniques.

• VivaSpin 15R filters: Useful secondary concentration

step in process and drinking water samples

• PEG precipitation was optimised.

Nucleic acid extraction• Acceptable recovery of bacterial DNA and viral RNA

extraction with NucliSENS®

• Contradictory results for protozoa when comparing

PCR and verification methods could be related to the

extraction method

(not effective for cysts and oocysts)

• Good tool in terms of recovery and concentration for viruses

and bacteria in process and drinking water with large water

volumes.

• Not useful for very loaded water sources for bacteria (i.e

Spanish site raw water). In addition. Humic substances in

water sources may cause excessive foam formation during

elution.

• Enabled verification analyses, such as Colilert®, to be more

sensitive, thus lowering the detection limits significantly.

• Not present any clogging problems in any of the sampling

campaign, even when filtering 1000 L (drinking water)

WP10-Large Systems

DNA extraction and detection (PCR)• Virus:

– Viruses were detected in the first treatment steps at Spanish and German site (surface water). No detection of virus in

Danish and UK Site 1 (ground water). Hepatitis A virus was detected for in Spanish site raw water.

– Punctual detections in low quantity values in distribution network

• Bacteria

– E.coli and Campylobacter spp have worked properly. Detection in raw water and reduction through treatment observed.

– A few low positive detections by qPCR in distribution network, mostly negative for verification methods.

• Protozoa

– 1 punctual positive detection in Spanish site DN, while verification result was under detection limit. Inconsistent with the

hypothesis that the nucleic acid extraction is not working properly for protozoa.

Lessons learnt? Where, when and how to use

the new techniques

• Results of microscopy and FISH-based techniques have shown reduction through DWTP treatment with the parameters total cell

counts, viable cell count and thermophilic Campylobacter

• E.coli FISH detection has been inconsistent sometimes, maybe due to particles in the concentrate

• The FISH-based microscopic analyses of total and viable cell counts have brought new information about dead/live bacteria ratio

in the DWTP and DN.

• Campylobacter were detected in two cases in drinking water, which were not in line with verification methods and PCR results.

False positives because of unspecific hybridisation.

Detection (FISH)

WP10-Large Systems

BACTcontrol Conclusions

E.coli / coliforms module

• The enzymatic activity of total coliforms in raw water, measured by

BACTcontrol, correlated very well with the cultural verificationmeasurements.

Total Activity module

• Drinking Water Treatment Plants

– BACTcontrol Total Activity A is a robust online system to monitor

viable bacteria.

– Total activity is an interesting tool to monitor the impact of quality

events involving microbiological risk and to check the performance of

disinfection steps, as seen in TW and GAC study cases.

• Distribution Network

– Very low TA values have been found during all validation period.

Lessons learnt? Where, when and how to use

the new techniques