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5/13/2015 The Paul L. Busch Award Recognizing Significant Advances in Water Quality Research Wednesday, May 13, 2015 2:00 ‐ 3:30 pm ET How to Participate Today • Audio Modes • Listen using Mic & Speakers • Or, select “Use Telephone” and dial the conference (please remember long distance phone charges apply). • Submit your questions using the Questions pane. • A recording will be available for replay shortly after this webcast. 1

5/13/2015 Today’s Moderator Amit Pramanik, Ph.D., BCEEM Director of Research apramanik@werf.org 571 ‐ 384 ‐ 2101 Today’s Agenda 2:00 pm Welcome and Introductions Doug Owen / Amit Pramanik 2:10 pm Nexus of Water Sustainability and Public Health: Antibiotic Resistance in Recycled Water Amy Pruden 2:40 pm The Interplay Between Chemicals and Microorganisms in Urban Water Systems Nancy Love 2:55 pm Engineered Platforms and Pathways for Resource Recovery from “Waste” Kartik Chandran 3:10 pm Engineering Better Biofilms: Rational Design of Attachment Surfaces to Improve Their Performance Andrew Schuler 3:25 pm Panel discussion / Q&A All 3:40 pm Adjourn 2

5/13/2015 Douglas Owen, P.E., BCEE, ENV SP ARCADIS WERF Board of Directors Paul L. Busch Award “WERF’s goal of developing the scientific understanding and the technology which will improve the environment in a sustainable manner is a goal which everyone in our profession can share.” – Paul Busch 3

5/13/2015 The Paul L. Busch Award • 2001 ‐ NANCY LOVE • 2010 ‐ KARTIK CHANDRAN • 2002 ‐ LUTGARDE RASKIN • 2011 ‐ VOLODYMYR TARABARA 2003 ‐ DAVID SEDLAK 2012 ‐ ROBERT NERENBERG • • • 2004 ‐ BRUCE LOGAN • 2013 ‐ CHUL PARK • 2005 ‐ DANIEL R. NOGUERA • 2014 ‐ AMY PRUDEN • 2006 ‐ PAUL WESTERHOFF To learn more, go to: • 2007 ‐ PAIGE NOVAK http://www.werf.org/i/Awards/a/Awards 2008 ‐ ANDREW SCHULER • /Awards.aspx • 2009 ‐ JAEHONG KIM Click on “The Paul L. Busch Award” link Amy J. Pruden, Ph.D. Kartik Chandran, Ph.D. Virginia Tech Columbia University Nancy G. Love, Ph.D., P.E., BCEE Andrew Schuler, Ph.D. University of University of New Mexico Michigan 4

5/13/2015 Amy J. Pruden, Ph.D. Virginia Tech 2014 Paul L. Busch Award Recipient Nexus of Water Sustainability and Public Health: Antibiotic Resistance in Recycled Water Paul L. Busch Award 2014 Amy Pruden Professor Image: Rodney M. Donlan, CDC Via Department of Civil & Environmental Engineering 5

5/13/2015 Human Progress: Our Water Infrastructure Our Water Infrastructure Picture Credit: Marc Edwards Images: Tracey Saxby, IAN Image Library; Ixnayonthetimmay; Leo 'Jace' Anderson ‐ FEMA 6

5/13/2015 Recycled Water INDIRECT • Need for Water Sustainability • Direct and Indirect Potable Reuse DIRECT • Nonpotable Reuse • Role of bacterial regrowth for microbial constituents of emerging concern – Opportunistic Pathogens (OPs, e.g., Legionella ) – Antibiotic Resistance Genes (ARGs) NON ‐ POTABLE In addition to access 1900 to clean water, antibiotics are largely responsible for the high quality of life we enjoy today 1997 Bacterial infections CDC, MMWR, July 30, 1999 / 48(29);621 ‐ 629 7

5/13/2015 Sustainability of Antibiotic Use Spellberg, B. 2010. Spellberg, B. 2010. I NCREASED RESISTANCE D ECREASED A NTIBIOTIC D EVELOPMENT 15 Antibiotic Resistance in the U.S. • September 2013 CDC Report: • 2 million Americans fall ill from antibiotic ‐ resistant bacteria • At least 23,000 die as a result (many more if count complications) • Community ‐ acquired MRSA now surpasses hospital ‐ acquired MRSA “ Antibiotic-resistant infections can happen anywhere. Data show that most happen in the general community ” 8

5/13/2015 Antibiotic Resistance Genes (ARGs) as Environmental Contaminants Antibiotic Resistance ARG Transposon Mutations in chromosome Plasmid Mobile DNA Integron genetic elements (Antibiotic Resistance Genes or ARG) Artistic Credit: Heather Chromosomal Storteboom DNA Horizontal Gene Transfer (HGT): Traditional approach of killing bacteria may not be sufficient- ideally should think about destroying ARGs. 9

5/13/2015 Antibiotic Resistance Genes (ARGs) Selective Selective Pressure Pressure Run off Water Reuse ARGs Correlate with Animal Feeding Operations and WWTPs in the Poudre River Watershed (R 2 =0.92, sul 1 p<0.001)! Pruden et al. ES&T 2012 10

5/13/2015 Microbial Ecology in Pipe Biofilms Additional Parameters Selective Contributing to Pressure Regrowth: Necrotrophic Growth  Decay of Transduction Disinfectant Uptake by Residual Amoeba Horizontal  Stagnation Conjugation Gene  Elevated Transfer Selective nutrient (C,N,P) Predation Natural concentration Transformation  Temperature Amoeba encystment Extracellular Polymeric Substances Artistic Credit: Emily Garner Reclaimed 10 6 10 6 erm F gene copies/mL tet (A) gene copies/mL erm F tet (A) 10 5 10 5 Water 10 4 10 4 Pipes 10 3 10 3 10 2 10 2 ∙ Most ARGs detectable 10 1 10 1 at the point ‐ of ‐ use , but tet (O) gene copies/mL 10 6 10 6 sul 1 gene copies/mL tet (O) not exiting treatment sul 1 10 5 10 5 plant 10 4 10 4 ∙ van A detectable 10 3 10 3 throughout 10 2 10 2 10 1 10 1 ∙ Highlights importance 10 6 sul 2 gene copies/mL 10 6 of considering the sul 2 van A gene copies/mL van A 10 5 10 5 microbiology that 10 4 10 4 happens as water flows 10 3 10 3 through pipes 10 2 10 2 Fahrenfeld et al . 10 1 10 1 Frontiers in Aa-POE Ab-1POE Ab-2POE A-2POU A-5POU A-8POU A-1POU A-3POU A-4POU A-7POU A-6POU Aa-POE Ab-1POE Ab-2POE A-2POU A-5POU A-8POU A-1POU A-3POU A-4POU A-7POU A-6POU Microbiology 2013 11

5/13/2015 Fahrenfeld et al . Frontiers in Microbiology 2013 Irrigating Soil Slurries with Reclaimed Water sul 1 sul 2 tet (O) tet (W) Potential Mitigation Endpoints • Comparable to a defined control background – ARG diversity – ARG abundance – Absence of key clinical ARGs (e.g., NDM ‐ 1) – All of the above: HGT/multi ‐ drug markers 12

5/13/2015 Objective: Paul L. Busch Award • Compare ARGs in reclaimed and potable water distribution systems – Potable water is an important “control” – Potable water distribution system management can inform distribution of recycled water – Examine role of microbial re ‐ growth – Use next generation DNA sequencing for deep insight into microbial community and ARGs – Compare with culture ‐ based methods Distribution System Survey Biofilm Sample Collection Bulk Water Water Chemistry – pH, temperature, – Before treatment disinfectant residual, – Point of Entry DO, turbidity, metals, – 5 Dist. System (Point of Use) anions 13

5/13/2015 Microbiology Methods OPs ARGs Illumina HiSeq qPCR E. coli and Enterococcus resistance profiles Minimum Inhibitory Concentration Enterolert / Colilert Overview of Systems POTABLE RECLAIMED WATER WATER System Disinfectant Summary of Treatment Disinfectant A Cl 2 Plant #1 – Advanced wastewater treatment- Cl 2 (ClNH 2 Bardenpho Process Residual) Plant #2 – Activated sludge, secondary clarification, denitrification B Cl 2 ; Plant #1 – Advanced wastewater treatment Cl 2 occasional – Bardenpho Process; UV ClO 2 Plant #2 – Biofiltration, secondary (ClNH 2 sedimentation Residual) C Cl 2 Dual media filters or Cl 2 membrane bioreactors (ClNH 2 Residual) Note: All potable water sources are a combination of surface and groundwater 14

5/13/2015 Microbial Community: Illumina Amplicon Sequencing Group by: Utility and Type Group by: Water Type So A: Reclaimed B: Reclaimed C: Potable A: Potable C: Reclaimed B: Potable UNIFRAC Weighted Microbial Community: Illumina Amplicon Sequencing Group by: Utility Group by: Water Type So C B A UNIFRAC Weighted 15

5/13/2015 Microbial Community: Illumina Amplicon Sequencing Group by: Matrix Group by: Water Type Biofilm Bulk Water UNIFRAC Weighted Microbial Community: Illumina Amplicon Sequencing Group by: Water Type Potable Reclaimed R statistic: 0.492 p: 0.1 % UNIFRAC Weighted 16

5/13/2015 Water Chemistry as a function of Water Age *Disinfectant Residual: Cl 2 Impact of Water Chemistry on OPs 17

5/13/2015 Regrowth of OPs Utility A: Potable (Residual: ClNH 2 ) Utility A: Reclaimed (Residual: Cl 2 ) Bulk Water Bulk Water POE 1 2 3 4 5 POE 1 2 3 4 5 [log (copies / mL)] [log (copies / mL)] 16S rRNA 4.0 3.3 3.6 4.1 4.8 6.2 16S rRNA 5.4 6.6 6.4 6.8 6.6 7.3 Legionella spp. 2.1 3.2 Legionella spp. 4.0 3.8 3.2 4.0 4.4 L. pneumophila L. pneumpohila 3.0 L. pneumophila L. pneumpohila 2.6 2.6 2.7 2.6 3.0 Mycobacterium spp. 2.6 3.4 Mycobacterium spp. 2.5 2.6 2.7 2.8 2.5 M. avium 2.0 1.8 1.8 M. avium N. fowleri N. fowleri 2.5 Acanthamoeba spp. 3.0 Acanthamoeba spp. 2.5 V. vermiformis 1.0 1.8 V. vermiformis Biofilm Biofilm 16S rRNA 4.1 3.9 4.0 3.9 4.4 16S rRNA 5.0 4.9 5.0 4.4 5.0 [log (copies / cm 2 )] [log (copies / cm 2 )] Legionella spp. 3.3 2.7 Legionella spp. 3.4 3.4 3.4 3.3 3.1 L. pneumophila L. pneumpohila 2.8 2.8 L. pneumpohila 2.7 3.4 3.7 2.5 2.8 L. pneumophila Mycobacterium spp. 3.1 2.7 2.7 3.0 Mycobacterium spp. 3.1 M. avium M. avium N. fowleri 3.0 N. fowleri Acanthamoeba spp. Acanthamoeba spp. V. vermiformis 2.4 V. vermiformis Metagenomics: Average ARG Composition 18

5/13/2015 Metagenomics: ARGs in Reclaimed Water Metagenomics: ARGs in Reclaimed Water vs. Potable Water : Reclaimed 19