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Second generation of rapid testWhat you need to know about ATP 2G and one-hour tests on fields for Legionella Risk Assessment
Monthly Analysis– Slow increasing
Weekly Analysis– High Variability– Trend to more stability
janvier février mars avril mai juillet
D’après le syprodeau: plate frome legionelle 2008
Biofilm
Amoebae
Ciliés
Legionella inside granule
Legionellainside Kyste
amoeba
LegionellaViable Non
culturable (VBNC)
Permanent release ofgranules from ciliates
Deadlegionella
LégionellaViable
cultivables (VC)
Biofilm, amoeba vesicles, granules ciliates, amoeba cysts: resistant to disinfectionLegionella can proliferate within free-living amoebae (FLA) where they are protected from disinfectants
Different form of Legionella life
Legionella treatment – Selection of Legionella Life Relevance of information
Légionella VC
LégionellaVNC
Dead
Legionellainside amoeba
Legionella from ciliatesLégionella inside Ciliates
LégionellaInside kyste
Biocide
Selection of Resistance of legionella form
No effect
More Pathogens+ +++
Methods for Legionella risk assessement
PCR
culture
Total Flora
Bacteria(Legionella , total flora)
AlguaeProtozoar
VBNCVC…
ATP= Microbial activityRevivable Flora
Legionella
Anaerobic
Aerobic22°C
Aerobic36°C
Microorganisms have ATP …
Specific method
Légionella
Specific Legionella Indicator
PC
R
?
?
VC
VBNC
Dead
Intra amoeba/ciliates
Legionella inside granule
?
Culture
v PC
R
?
Difficult correlation between all these different approach
Rel
evan
ce a
naly
sis
LegionellaViable Non
culturable (VBNC)
LégionellaViable
cultivables (VC)
Amoebae
Permanent release ofgranules from ciliates
New generation ATP-metry method - ATP2G
Method for simple and fast measurement of ATP (Adenosine TriPhosphate) concentration in the cell
2Nd generation dedicated to water samples
Adenosine Triphosphate ( ATP)Issued from the Glycolyse reaction in living cells, ATP is the energy source for many chemical reactions Provides the energy necessary for all biological functions
• Adaptation to the environment, Food consumption, Reproduction
ATP is an essential molecule for microbial lifeAerobic, anaerobic and anoxic bacteria
How does the ATP technique work? The technology is based on bioluminescence principleAn enzymatic reaction transforms ATP in Light, in the presence of Luciférine / Luciférase complex
Primary result delivered in Relative Light Units ( RLUs)
Linear relationship between RLUs and ATP concentration
ATP + luciferin + O2 AMP + PPi + oxyluciferin + LIGHTMg++
luciferase
ATP G2 vs. 1st Generation ATP Testing
• Quantitative sample transfer ensures accuracy
• Higher volume analyzed – More representative
• Superior chemistry of reagents
• Optimized protocols ensure minimal interferences (TDS, TSS, Oil, Biocides)
• Liquid-stable ATP standard (UltraCheck 1) converts RLU to quantitative concentration
• Different test kits optimized for different applications
Significance of cATP measurement
11
Analysis of Legionella risk in water
Accepted by regulationCulture method
Introduction of nutrients + bacteria
Growth of biofilm
Establishement of ecosystem favorable
for Legionella
Proliferation of Legionella in biofilm
Proliferation of Legionella in water
Above regulatory thresholdsPlanktonic Legionella acquire
cultivability capacity
Microbiological risk
Potable water
Protocol
-> elimination of extracellular ATP
-> Lysing solution
pg ATP/ml ou Equivalent microorganisms
1- sample filtration 2- Extraction of Intracellular ATP 3. Analyse RLU cATP
QGA – significance of cATP value
Quantification of all living microorganisms through Intracellular ATP
Culturable, Viable but non culturablemicroorganismsAerobic, anaerobic, anoxic microorganismsBacteria , protozoa, algae,
True value of Total living flora in a sample
Stressed or VBNC
VC
QGA kit – WHY?
High value inspection of water installationsMicrobial risk analysis– potable water, sanitary water, cooling towers, industrial process waterLocalization of critical zones with biofilm build-upDetection of microbial disordersQuantification of circulating biomassEvaluation of efficiency of treatments (oxidizing, organic or physical)
High throughput screeningSelect critical points for regulatory analysis ( eg. Total flora, Pseudomonas aeruginosa or Legionella by culture or PCR)
ATP / culture on GVPC
ATP allows surveillance of biomass growth in water and biofilmsLegionella is a minority bacteria in microbiological ecosystem. Legionella pneumophila species is minority in Legionella groupGVPC culture : only the 36°C culturablefraction of Legionella after 10 days of growth.This is the regulation method
Flow cytometryFlow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid – Total Cell Count (TCC)
Counts all microorganismsNot linked to viability 2h to results
Total flora count using QGA kit
Measurement of all viable microorganisms
Comparison of HPC, TCC and ATP-metry
Data from one representative tap over the day
TCC and ATP: R2 = 0,97TCC and HPC: R2 = 0,72TCC and ATP: R2 = 0,56
TCC and ATP are more sensitive and accurate representation of water qualityTrends of all 3 methods are the same
Overall correlation between TCC and ATP
R2 = 0.69
ATP and TCC correlate very well together
Overall correlation between ATP-metryand HPC
R2 = 0.31
HPC and ATP don’t correlate well - only one small part (1%) of living bacteria cultivate on
selective media
TCC and ATP are rapid and more sensitive methods for water quality monitoring than HPC
TCC and ATP detect all microorganisms without taking into account their cultivability
ATP-metry can be performed on the field for Bio audit -
A routine monitoring program a part of water management plan
Monitor raw water quality to assess treatment in real-timeMeasure true total living microorganismQuickly audit a water distribution/treatment system to locate problem areas.Optimize any disinfection program (eg chlorine, chloramines) to find the optimum point between microbiological contamination and disinfection by-products (DBP’s).Monitor water quality in reservoirs and at booster stations to optimize system management.Control biocide usage to lower operating costs and minimize public health risk.Manage maintenance programs including line flushing, well flushing, cross-connections and line breaks!Assess / manage customer complaints
BioAUDIT by USING ATP INDICATORCase of LEGIONELLA
PNEUMOPHILA Medical retirement home
L. NEYRAT, L. BELOTTI, C. HERNANDEZ, J. FOEGLE, Th. LAVIGNEHôpitaux Universitaires de Strasbourg
Water Network
Biological mapping
Volume of water cATP
Cold water city 17371 65 50 0,7484 14,5Cold water city 17371 64 50 0,7369 14,5Hot Water departure at Heat exchanging 17371 3290 50 37,88 60,8Hot Water departure at Heat exchanging 17371 2645 50 30,45 60,8Hot Loop back 17371 4522 50 52,06 51Hot Loop back 17371 3206 50 36,91 51
Temperature °CSampling points UltraCheck RLU
ATP RLU
Colum Number Point of sampling Localisation Pg ATP /ml Temperature °C1 18 RDC Col. 1 EF 16,8°C Ground floor Cold water 1,382 16,81 20 2ème Col. 1 EF 21°C 2nd Floor Cold water 0,7599 211 23 4ème Col. 1 EF 24,8°C 4 floor Cold water 1,094 24,8
12 26 2ème Col. 12 EF 13,2°C 2nd Floor Cold water 0,7944 13,212 27 4ème Col. 12 EF 13,2°C 4 floor Cold water 0,7599 13,211 30 RDC Col. 11 EF 13,4°C RDC Cold water 1,266 13,411 32 2ème Col. 11 EF 14,6°C 2nd Floor Cold water 0,7829 14,611 34 4ème Col. 11 EF 14,1°C 4 floor Cold water 1,347 14,116 49 RDC Col. 16 EF 13,2°C Ground floor Cold water 1,43 13,216 43 2ème Col. 16 EF 14,3°C 2nd Floor Cold water 1,071 14,316 36 4ème Col. 16 EF 15,3°C 4 floor Cold water 0,6793 15,315 51 RDC Col. 15 EF 17°C Ground floor Cold water 1,288 1715 52 RDC Col. 15 EF 59,4°C Ground floor Cold water 45,48 59,425 45 2ème Col. 25 EF 14,1°C 2nd Floor Cold water 1,314 14,125 38 4ème Col. 25 EF 15°C 4 floor Cold water 0,7484 1524 47 2ème Col. 24 EF 14,2°C 2nd Floor Cold water 0,7343 14,224 40 4ème Col. 24 EF 14°C 4 floor Cold water 1,692 1427 56 RDC Col. 27 EF 13,5°C Ground floor Cold water 0,7988 13,528 53 RDC Col.28 EF 13,5°C Ground floor Cold water 0,7601 13,5
Biological mapping cold water
cATP
pg/mL1 19 RDC Col. 1 EC 59°C Ground floor HOT WATER 37,72 591 21 2ème Col. 1 EC 59°C 2nd Floor HOT WATER 57,69 591 24 4ème Col. 1 EC 52°C 4 floor HOT WATER 40,45 52
12 26Bis 2ème Col. 12 EC 59°C 2nd Floor HOT WATER 56,07 5912 28 4ème Col. 12 EC 58,2°C 4th floor HOT WATER 23,22 58,211 31 RDC Col. 11 EC 59°C RDC HOT WATER 60,93 5911 33 2ème Col. 11 EC 59,2°C 2nd Floor HOT WATER 32,68 59,211 35 4ème Col. 11 EC 57,8°C 4th floor HOT WATER 30,88 57,816 50 RDC Col. 16 EC 59,3°C RDC HOT WATER 23,76 59,316 44 2ème Col. 16 EC 59,2°C 2nd Floor HOT WATER 33,86 59,216 37 4ème Col. 16 EC 59,4°C 4th floor HOT WATER 70,3 59,425 46 2ème Col. 25 EC 59,1°C 2nd Floor HOT WATER 24,43 59,125 39 4ème Col. 25 EC 58,3°C 4th floor HOT WATER 21,17 58,324 48 2ème Col. 24 EC 59°C 2nd Floor HOT WATER 18,17 5924 41 4ème Col. 24 EC 59°C 4th floor HOT WATER 12,9 5927 57 RDC Col.27 EC 57,5°C Ground floor HOT WATER 148,7 57,528 54 RDC Col. 28 EC 53,5°C Ground floor HOT WATER 109,1 53,5
Temperature °C
Colum Number
Sampling points
Biological mapping hot water
2nd biological mapping
cATP 3 Mai 2013
cATP 24 Mai 2013
pg/mL pg/mL
1 19 RDC Col. 1 EC 59°C Ground floor 37,72 59 0,9 65,71 24 4ème Col. 1 EC 52°C 4 floor 40,45 52 0,3 63,3
12 28 4ème Col. 12 EC 58,2°C 4 floor 23,22 58,2 1,2 67,211 31 RDC Col. 11 EC 59°C Ground floor 60,93 59 0,2 66,511 35 4ème Col. 11 EC 57,8°C 4 floor 30,88 57,8 0,7 64,816 50 RDC Col. 16 EC 59,3°C Ground floor 23,76 59,3 0,2 65,316 37 4ème Col. 16 EC 59,4°C 4 floor 70,3 59,4 1,2 66,525 39 4ème Col. 25 EC 58,3°C 4 floor 21,17 58,3 0,04 61,524 41 4ème Col. 24 EC 59°C 4 floor 12,9 59 0,9 64,727 57 RDC Col.27 EC 57,5°C (filtr Ground floor 148,7 57,5 1,1 67,128 54 RDC Col. 28 EC 53,5°C Ground floor 109,1 53,5 0,3 51,2
Column N°
Sampling point Temperature °C 3 May 2013
Temperature °C 24 May 2013
Conclusion
Immediate action network of global improvement were considered preferable to the prohibition to use sinks- Separation of the hot water network from the building renovation- Increasing number of flush - Temperature increase production of domestic hot water
Study of the total flora present in the water system by ATP 2G correlated well in this example the results of the microbiological samples, with the advantage of timeliness and the possibility of large series of analyzes (More then 60 point of sampling ).
GENERAL CONCLUSION
Quantitative ATP-metry can be used as a complementary technique to the culture-based method for Investigation .
This technique presents several advantages:– This technology allows a real-time on-site diagnostic of the
network (3 minutes per sample) – A low-cost diagnostic in comparison to the standard
analytical technique (15 to 20% more expensive for a legionella analysis);
– This technique increases the number of analyzed samples; The ATP 2G allows the identification and the monitoring
of the critical control points in a water network.
