1. Biology Program, FDEP Laboratory Evaluation of PMA-qPCR for Quantitative Differentiation of Live Human-associated Bacteroidales for Water Quality Monitoring Puja Jasrotia, Daisys Matthews, Loretta Wolfe and David Whiting May 18, 2016

2. Outline Microbial Source Tracking (MST) toolbox approach Study objectives Lab standardization Field testing Summary Future directions 5/18/20162

3. MST Toolbox Approach 5/18/2016 3

4. Study objectives Validate and incorporate new tools for microbial source identification for water quality monitoring Examine the suitability of the Propidium Monoazide (PMA) method and incorporate it into FDEP Laboratory’s current molecular source markers 5/18/20164

5. Propidium Monoazide Treatment PMA is a photo reactive dye. Amplifies only intact cells in qPCR (live vs. dead) Intercalates with dsDNA in membrane compromised dead cells Forms stable covalent bond upon photolysis Detection by qPCR will not amplify PMA modified DNA, hence permitting selective detection of live cells Potential to distinguish between recent and past fecal contamination 5/18/20165 Source: PMA TM mechanism of action (PMA TM product information; www.biotium.com)

6. 5/18/2016 6 Grow B. dorei Analyze results Check viability Lab standardization (B.dorei pure culture) PMA con (mM) Heat killing temp ( 0 C) Heat killing time (min) PMA incubation (min) Light exposure (min) 50, 75, 10072, 80, 8510, 15, 205, 10, 2010, 15, 20 Treatment Parameters Tested Spike PBS with B. dorei Filter sample B. Heat killed A. Live DNA extraction and qPCR +/- PMA treatment C. 1:1 mix of live and heat killed cells

7. Lab standardization (B.dorei pure culture) 5/18/20167 qPCR (total DNA) - similar recovery across treatments PMA-qPCR - ~3 log decrease in heat killed Heat-killed cells had a 13 Ct difference with PMA

8. Field testing Wastewater treatment plant (WWTP) 5/18/20168 WWTP Influent/ Effluent Dilute with PBS Filter 100 mL sample B. Heat killed A. Live DNA extraction and qPCR +/- PMA treatment C. 1:1 mix of live and heat killed cells

9. Field testing (WWTP influent) 5/18/20169 Influent results similar to B.dorei culture results No DNA recovery in effluent

10. Field testing (WWTP Influent) 5/18/201610 WWTP Influent Dilute with PBS Filter 100 mL sample DNA extraction and qPCR +/- PMA treatment Hold time (0,24,30,48 hr)

11. Field testing (Holding time – WWTP influent) 5/18/201611 Influent stored in refrigerator and diluted at each time interval Average ΔCt increases with hold time, demonstrating loss of viability over time

12. Field testing Surface water urban creeks near St. Johns River, Jacksonville, Florida Hogan creek (sites A2 and A3) Miller creek Verified impaired for fecal coliform Sampling: January 12, 2016 Parameters: qPCR – HF183 Micro – E.coli, Enterococci Chemical – Sucralose, Acetaminophen 5/18/201612

13. Results 5/18/201613 High Acetaminophen – potential untreated wastewater source High Sucralose – potential wastewater source High E.coli and Enterococci MPNs – potential fecal pollution Low level detect with HF183 marker Sampling sites Acetaminophen (ug/L) Sucralose (ug/L) E.coli (MPN/100 ml) Entero (MPN/100 mL) HF183-Bacteroidales Without PMAWith PMA ΔCt CtQTY (GEU/100ml)Ct QTY (GEU/100 ml) A2 Hogan Creek from Culvert 1.40.4103568829.86 3996 35.121445.26 A3 Hogan Creek from open ditch 0.004 (U)0.07124201553No detect Miller Creek @ Shumacher Ave. 0.004 (U)0.0982420 35.8786No detect

14. Summary PMA-qPCR is an effective method to distinguish live from membrane-compromised cells Promising results from this study: Determined appropriate conditions for using PMA-qPCR on bacterial cultures and applied them to field samples Consistent results in culture and wastewater samples Impaired environmental water samples showed low levels of detection of HF183 after PMA treatment 5/18/201614

15. Future directions Additional sampling needed to characterize use of PMA in MST studies and water quality monitoring Statistical analyses of data and relation to fecal indicators Development and validation of PMA-qPCR with additional MST markers 5/18/201615

16. Contact Information Puja Jasrotia, PhD Environmental Consultant, Biology Program Puja.Jasrotia@dep.state.fl.us (850) 245-8175 Contact Information Puja Jasrotia, PhD Environmental Consultant, Biology Program Puja.Jasrotia@dep.state.fl.us (850) 245-8175 5/18/201616 Questions?