3. Future ?
E. coli Enterococcus faecalis
EC 2006**
E. Coli (500/100mL) Feacal enterococci (200/100mL)
USEPA 1986*
Escherichia coli (235/100mL) Feacal enterococci (104/100mL)
EEC 1975
Total coliform (10,000/100mL) Feacal coliform (2,000/100mL) Feacal streptococci (100/100mL)
* Based on Geometric mean
** Based on 95% evaluation

4.  The most common enterococci in human feces (90-95%)
 Responsible for the majority of human enterococcal
infections
 High endogenous resistance to stressful
conditions by activating the VBNC state
 High ability to acquire and transfer rapidly
various resistance genes

5. (EPA 1600)
37 ± 0.5°C 24h
• Long incubation time
• Underestimating methodologies:
1. VBNC bacteria are not counted
2. Heterogenic distribution (plates)
3. Neighboring colonies dynamics (plates)

6. TaqMan® probe techniqueSYBR® Green technique

7. Primers used: 16s rDNA gene, DNA of E. faecalis v583

8. Gene
selection
• Scientific literature
• Database search and analysis (NCBI, RDP etc.)
QPCR
system
design
• Short amplicons (50-150bp)
• Dimers and steady structures formation
• Hair pin loops and amplicon mfold
• Base pair composition – Tm adjustment and fluorophore quenching
Systems’
screening
• Specificity tests
Accuracy and
precision
•Spiking sterile seawater samples with known quantities of bacteria
•Environmental samples processing and comparison with the standard methods’ quantification
Protocol
development
• Concentrating bacteria from seawater
• DNA extraction
• Removal of inhibitory substances and PCR inhibition control
• Dead bacteria screening
High efficiency and sensitivity

9. Species
Amplification
16s rDNA PBP5 GroES
Phylum "Firmicutes”
Class "Bacilli"
Order "Lactobacillales"
Genus Enterococcus
E. faecalis V583 + + +
E. faecalis 14506 + + +
E. faecalis 2020 + + +
E. faecalis 29212 + + +
E. faecalis F76-117 + + +
Genus Enterococcus E. faecium ISO - - -
E. faecium 180 - - -
Order "Lactobacillales" Aerococcus viridans - - -
Class "Bacilli" Bacillus sp. - - -
Staphylococcus aureus - - -
Staphylococcus epidermidis - - -
Gastrointestinal and water
quality standard bacteria
E. coli K12 MG1655 - - -
E. coli K12 Hfr - - -
E. coli 8A - - -
E. coli 7L - - -
E. coli 7G - - -
E. coli environmental isolate - - -
Gastrointestinal bacteria Shigella soneii + + +
Salmonella enterica - - -
Citrobacter freundii - - -
Serratia marcescens - - -
Enterobacter cloaca - - -
Klebsiella oxytoca - - -
Marine bacteria Seawater isolate #1-3 - - -
Env. Vibrio sp. #1, #4 and #5 - - -
Vibrio shiloi - - -
Vibrio corallilyticus - - -
Vibrio harveyi CM18 - - -
Vibrio harveyi CM19 - - -
Vibrio harveyi CM32 - - -
Vibrio harveyi CM37 - - -

10. 16s rDNA PBP5GroES
Amplification
without
E. faecalis
genomic DNA
Amplification
with ~3000
E. faecalis
Genomic
units/well
E. coli
S. aureus
Bacillus sp.
V. corallilyticus
V. Shiloi
Vibrio #1
SW #1
SW #2

11. The addition of the TaqMan probe resulted with 100%
specificity against the 36 tested strains
Small amount of target DNA is sufficient to eliminate the
production of all non-specific PCR products
The use of 3 amplification systems lowers the odds to
encounter another bacterium that have all sequences of
primers and probes placed in the same manner in its
genome

13. 16s rDNA
E. coli-sfmDGroES
PBP5
Criteria level
X10
X100
Criteria level
X10
X100
Criteria level
X10
X100
Criteria level
X10
X100

14.  Plate counts underestimation?
OR
 QPCR overestimation?
 GroES?
First assay

15. First assay Second assay

16. F(3,64)=0.83, P=0.48
One way ANOVA
No significant
difference
'between
methods
quantification
Contaminated
Contaminated
Non-
contaminated
Non-
contaminated
Contaminated
Contaminated
Non-
contaminated
Non-
contaminated
Different
French
beaches
QPCRMPN

17.  Molecular techniques, specifically nucleic acid amplification
procedures, provide sensitive, rapid and quantitative analytical
tools for the detection of specific organisms.
 Factor contributing for qPCR robustness:
- Detailed design of each amplification system
- Optimization of the full protocol
 Fundamental differences between amplification systems have
led for the design of a system-specific internal control.

18. Professor Adam Friedmann
Itay Kilovaty
Alon Daya
Dr. Tzachi Bar
Tal Amit
Marina Schetman
Professor Stella Mitrani-Rosenbaum

20. 16s rDNA
GroES
PBP5

21. ConclusionBetween
methods
Between
amplification
systems
Seawater samples
and treatment
Null hypothesis is correct
– no difference
F(3,64)=0.83
, p=0.48
F(2,48)=0.411
, p=0.66
Toulon samples
Null hypothesis is correct
– no difference
F(2,15)=0.41
9, p=0.66
F(1,10)=0.78,
p=0.4
Boulogne
samples
Null hypothesis is correct
– no difference
F(3,8)=0.09,
p=0.96
F(2,6)=0.006,
p=0.99
Boulogne –
improved DNA
extraction
Null hypothesis is correct
– no difference
---F(2,24)= 0.42,
p=0.66
Boulogne August
24-25 – improved
DNA extraction
*

22. QPCR
overestimation
VBNC
bacteria
Dead bacteria
QPCR
underestimation
Single
bacterium
quantification
Inhibition
Low DNA
yield