DEVELOPMENT OF RAPID QPCR APPROACHES FOR MEASUREMENT OF E. COLI AND ENTEROCOCCUS IN

ENVIRONMENTAL WATERS: THE FUTURE FOR ROUTINE MONITORING?

Rachel T. Noble, A. Denene Blackwood, and Seth YuNational Monitoring Conference

May 9, 2006UNC Chapel Hill Institute of Marine Sciences

Morehead City, NC

Bacterial indicator testing• Routine monitoring in US costs

>10 M USD annually• Majority of money spent in

sampling and analysis time• California alone responsible for

more than half of the monitoring in the US

• Aim: Protect public health • Impacts: Public perception,

economy, tourism, recreational water usage

• Need: Rapid, quantitative methods that can be used to accurately manage beaches and shellfish harvesting waters

Routine methods: bacterial indicators

• Total coliforms (TC) heterogeneous group composed of Escherichia, Citrobacter, Enterobacter, Salmonella, Shigella, Yersinia, and Aeromonas genera

• Fecal coliforms or E. coli: thermotolerant (44.5 C) subset of TC (FC can include Klebsiella), freshwater

• Enterococci, (> 20 species, e.g. faecalis, faecium, casseliflavus, durans, avium, gallinarum), marine waters

• Membrane filtration, Chromogenic substrate (IDEXX), and Multiple Tube Fermentation

• Require from 18-96 hours for results• Inaccurate management of recreational and

shellfish harvesting waters because of delay

The need for faster results

• Allow accurate management of beaches (keep open when clean and close immediately when not safe for swimming)

• Faster results = better tracking down sources of contamination

• Tracking sources down = reducing sources (or prioritizing reduction of human sources) = reduction of potential risks to the public

Immediate future: real world example

• 7:00 AM: Beach water sample collected• 8:00 AM: Return to lab, process using rapid

method and routine method (MF, MTF, IDEXX) • 10:00 AM: • 1) E. coli > 400/100 ml and/or Enterococcus

>104/100 ml or = Return and close beach, sample on periodic basis until clean

• 2) Enterococci <104/100 ml or E. coli < 400/100 ml= Keep beach open and sample again (or wait until next morning to repeat)

Longer term future

• Same time-scale (or maybe more rapid)• Measure pathogens instead of indicators (i.e.

conduct epidemiology studies)• Real time measurements on deployed systems

could provide hourly indications of water quality• Technology applicable to shellfish harvesting

waters, aquaculture effluents, stormwater runoff, NPDES permits etc.

Criteria for rapid methods• Time required for result• Threshold exceedance • Numerical result• Accuracy, Variability, Reproducibility• Portability• Specificity and usefulness of result for mitigation or

protecting public health• Training required• Data accessibility• Maintenance required of system (Deployable in situ,

unattended?)• Cost (initial buy in, and per sample)• Equivalent results to ‘classical’ indicator bacteria

data/historical data

Developing Rapid Detection Technologies for Environmental Waters

• Most currently developed technologies utilize sample collection and filtration approaches similar to routine methods

• Capture is dependent upon approach• Detection: fluorescence, electrochemical, etc.• Data transfer and real-time access• Combination of available applications limited only

by imagination and $$• Noble and Weisberg 2005

Capture: nucleic acid priming• PCR Based methods

• Transcription mediated amplification (TMA)

• Microarrays

• NASBA

• Highly specific/sensitive

• Based upon known sequence complementarity

• Can be used to type specific types of pathogens

Rapid microbial detection assays

• Developing technology using QPCR in conjunction with Cepheid, Inc.

1) QPCR- quantification of E. coli (~ 2.0 hr)2) QPCR- quantification of Enterococcus sp. (~ 2.0 hr)3) QRTPCR-quantification of human enteroviruses (~ 4 hr)4) QPCR and QRTPCR- Rapid assays for a wide range of

other viral pathogens (noroviruses, Vibrio vulnificus) and fecal marker bacteria (Bacteroides, Enterococcus species)

5) Apply technology in all types of water samples (estuarine, coastal, freshwater, brackish, wastewater, shellfish harvesting waters, shellfish harvesting meats)

QuantitativePCR (or RTPCR)

• Standard curve generated by plotting threshold cycle vs. log concentration and unknown values interpolated

• Increase in fluorescence is directly proportional to the amount of target cDNA in sample and indicated by cycle threshold

Unknowns

FluorescenceThreshold

Cyc

leT

hres

hold

Cepheid Smart Cycler II ®

• Primer/probe set design• PCR for amplification of DNA• No gels needed

QPCR Assay Details

• 100 ml through PC filter to capture bacteria• 0.45 µm pore size with vacuum needs same as for

MF analysis• DNA purification (bead beating or full extraction)• Assay features SmartMixTMTM Beads (all PCR

reagents) SmartBeadsTM TM contain primers, probes, and internal controls (lyophilized)

• Reduces pipetting steps and errors, interanalyst variability, increases quality of standard curves

• Assay also incorporates the use of innovative QPCR chemistry developed by DsX Limited (Manchester, UK) called SCORPIONS®

• Scorpions give lower background, lower LOD• Licensing fees not prohibitive in cost to WQ

agencies

Specificity and UbiquityProbe type tested

Organism ENT Scorpion I

E. coli Scorpion

Enterococcus faecium + -

Enterococcus faecalis + -

Enterococcus casseliflavus

+ -

Enterococcus hirae + -

Enterococcus gallenarium

+ -

Enterococcus pseudoavium

+ -

Enterococcus durans + -

Enterococcus dispar + -

Enterococcus malodoratus

+ -

Enterococcus raffinosus

+ -

Enterococcus mundtii + -

Probe type tested

Organism ENT Scorpion I

E. coli Scorpion

Escherichia coli JM 101 - +

Escherichia coli TB - +

Escherichia coli 8739 - +

Escherichia coli famp - +

Escherichia coli 2972 - +

Citrobacter - -

Bacteroides thetaiotaomicron

- -

Bacteroides fragilis - -

Vibrio parahaemolyticus - -

Vibrio vulnificus - -

Aeromonas hydrophila - -

Klebsiella oxytoca - -

Shigella flexneri and sonnei

- +

SCCWRP Study• June 2005 study to compare rapid detection methods for

Enterococcus and E. coli to routine methods (MF and either Enterolert™ or Colilert-18®)

• 3 day study• 54 blind samples • Seawater, stormwater runoff, blanks, coastal water unseeded

and seeded with cultured bacteria• Verification and speciation of isolates on plates and in

quantitrays• QPCR and TMA-based assays both fared well in the

comparison• Routine methods roughly 85-90 percent agreement• QPCR Enterococcus roughly 80% accurate• QPCR E. coli results 90% accurate• QPCR had low variability compared to other methods

E.coli by Colilert-18 Compared to QPCR y = 1.0004x

R2 = 0.8813

0

1

2

3

4

5

0 1 2 3 4 5

Log E. coli by QPCR

Lo

g E

. co

li b

y C

oli

lert

-18

X

X

1:1 line

E.coli by Colilert-18 Compared to QPCR: All Replicates

1

10

100

1000

10000

100000

1000000

1 10 100 1000 10000 100000 1000000

E. coli by QPCR

E.

coli

by

Co

lile

rt-1

8

Enterococcus Routine vs. QPCR: All replicatesy = 0.9179x

0

1

2

3

4

5

0 1 2 3 4 5

Log Enterococcus QPCR

Lo

g E

nte

roc

oc

cu

s m

EI

Enterococcus Routine vs. QPCR y = 0.9747x

R2 = 0.7135

0

1

2

3

4

5

0 1 2 3 4 5

Log Enterococcus QPCR

Lo

g E

nte

roc

oc

cu

s m

EI

Summary of results

• Fully quantitative, wide dynamic range• LOD for both assays is ~ 1 cell per reaction, for assay with DNA

extraction step is 10 cells/100 ml• 90% correct response rate for E. coli and 80-85% correct response

rate for Enterococcus• Strong correlation to routine methods (r2 values range from 0.70

to 0.95 depending upon comparison to MF or DS)• ENT assay captures at least 10 known species of Enterococcus

(including faecium, faecalis, caselliflavus, pseudoavium, gallinarum, etc.)

• Working towards ability to conduct filtration in the field• Internal controls and matrix controls in place for quantitative

sample analysis • Currently beta-testing assay at OCSD and exploring use in

drinking water (E. coli)

Preliminary results of beta testing at OCSD

• Roughly 40 samples• Enterococcus and E.

coli assay being tested by in house WQ personnel

• Results good to date• Open beaches and

storm samples• Stay tuned!

Enterococcus by mEI vs. Enterolert QPCR for All Samples as of March 15, 2006 using Standard Curve

Approachy = 1.0121x

R2 = 0.3578

012345

0 1 2 3 4 5

Log Enterococcus by mEI

Log

Ente

roco

ccus

Q

PCR

Acknowledgements:Steve Weisberg and John Griffith, SCCWRPDenene Blackwood and Jason Gregory (UNC Chapel Hill)State of California, esp. Robin McGraw and Shakoora Azimi-GaylonUnited States Department of AgricultureCepheid, Inc.: R. Schaller, C. Wilkins, N. Beckwith and S. YuOrange County Sanitation DistrictRich Haugland, USEPA