Fate of Antibiotic Resistance Genes During Anaerobic Digestion of Wastewater Solids Jennifer Hafer Miller Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Civil Engineering Amy Pruden William R. Knocke John T. Novak Diana S. Aga February 13, 2014 Blacksburg, Virginia Keywords: thermophilic anaerobic digestion, mesophilic anaerobic digestion, biosolids, pasteurization, MRSA, Escherichia coli, antibiotic resistance genes, ARGs, nanosilver, antibiotics, sulfamethoxazole Fate of Antibiotic Resistance Genes during Anaerobic Digestion of Wastewater Solids Jennifer Hafer Miller ABSTRACT Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies. This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur. Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone. Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer. Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs. iii DEDICATION This work is dedicated to my children: Catlin, Holland, and Baby Miller #3. I hope in some small way this research will contribute toward a healthier and safer environment for you. ACKNOWLEDGEMENTS This work was supported by U.S. Environmental Protection Agency STAR (Science to Achieve Results) Grant R834856, National Science Foundation Chemical, Bioengineering, and Transport Systems CAREER award #0852942, Virginia Tech Institute for Critical Technology and Applied Science seed funding and award TSTS 11–26, and Water Environment Research Foundation (WERF) Contract U1R12. Jennifer Miller was supported by the Charles E. Via, Jr. Department of Civil and Environmental Engineering Via Scholarship, Virginia Tech Graduate School Cunningham Fellowship, and WERF U1R12. I would like to express my sincere gratitude to Dr. William Knocke and Dr. Amy Pruden for your technical expertise, financial support, and the extreme level of patience and understanding that you have shown for my two "extracurricular" activities. I am also appreciative of my committee as a whole, Dr. John Novak and Dr. Diana Aga. You have been valuable technical resources and exemplary examples of academic professionals. My sincere thanks to Julie Petruska, Jody Smiley, and the Pruden Lab Group for all the help, tutoring, troubleshooting, and most of all friendship. I would like to acknowledge my parents, and their never-ending supply of interest, concern, love, and, of course, energy for babysitting. Last, but certainly not least, I would like to acknowledge my friend and husband - Jesse. These last five years have been the most challenging of my life emotionally, physically, and mentally - and school has been tough, too! I could not have done it without our combined blood, sweat, and tears. iv TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................... iv TABLE OF CONTENTS .....................................................................................................v TABLES ........................................................................................................................... vii FIGURES .......................................................................................................................... vii ATTRIBUTION ...................................................................................................................x CHAPTER 1: INTRODUCTION ........................................................................................1 Background .............................................................................................................1 Research Questions .................................................................................................3 Annotated Dissertation Outline ...............................................................................4 References ...............................................................................................................6 CHAPTER 2: INACTIVATION OF MRSA AND ESCHERICHIA COLI IN VARIOUS SLUDGE TREATMENTS: IMPLICATIONS FOR PATHOGEN AND ARG LOADING IN LAND APPLICATION OF BIOSOLIDS ...........................................................................9 Keywords ................................................................................................................9 Introduction .............................................................................................................9 Materials and Methods ..........................................................................................12 Results ....................................................................................................................18 Discussion ..............................................................................................................21 Conclusions ............................................................................................................27 Acknowledgements ................................................................................................28 References ..............................................................................................................28 CHAPTER 3: MECHANISMS OF ARG PERSISTENCE AND ATTENUATION DURING ANAEROBIC DIGESTION ............................................................................................. 48 Keywords ...............................................................................................................48 Introduction ............................................................................................................48 Materials and Methods ...........................................................................................51 Results ....................................................................................................................54 v Discussion ..............................................................................................................58 Conclusions ............................................................................................................61 Acknowledgements ................................................................................................62 References ..............................................................................................................62 CHAPTER 4: EFFECT OF AG NANOPARTICLES AND ANTIBIOTICS ON ANTIBIOTIC RESISTANCE GENES IN ANAEROBIC DIGESTION..................................................71 Abstract ..................................................................................................................71 Keywords ...............................................................................................................72 Introduction ............................................................................................................72 Methodology ..........................................................................................................75 Results ....................................................................................................................78 Discussion ..............................................................................................................81 Conclusions ............................................................................................................86 Acknowledgements ................................................................................................87 References ..............................................................................................................87 CHAPTER 5: ARG INCREASE IN RESPONSE TO COLD TEMPERATURE EXPOSURE: IMPLICATIONS FOR WINTER STORAGE OF SLUDGE AND BIOSOLIDS. ...........96 Significance and Impact of the Study ....................................................................96 Introduction ............................................................................................................96 Results and Discussion ..........................................................................................98 Materials and Methods .........................................................................................101 Acknowledgements ..............................................................................................102 References ............................................................................................................102 Supporting Information ........................................................................................108 CHAPTER 6: CONCLUDING REMARKS AND ENGINEERING SIGNIFICANCE ..............................................................................................................113 References ............................................................................................................115 vi TABLES Table 2-1. Primer sequences and annealing temperatures for QPCR assays. ....................17 Table S5-1. Probability values for t tests .........................................................................109 FIGURES Figure 2-1. Plate counts (CFU/ml PBS), ARG (mecA) and MRSA-specific gene (nuc) (gene copy numbers (gcn/ml PBS) for MRSA at 20 °C in PBS ......................................33 Figure 2-2. Plate counts (CFU/g TS), ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) for MRSA at 20 °C in digested sludge .........................34 Figure 2-3. Plate counts (CFU/ml PBS), ARG (mecA) and MRSA-specific gene (nuc) (gene copy numbers (gcn/ml PBS) for MRSA at 37 °C in PBS ......................................35 Figure 2-4. Plate counts (CFU/g TS), ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) for MRSA at 37 °C in digested sludge .........................36 Figure 2-5. Plate counts (CFU/ml PBS), ARG (mecA) and MRSA-specific gene (nuc) (gene copy numbers (gcn/ml PBS) for MRSA at 53 °C in PBS ......................................37 Figure 2-6. Plate counts (CFU/g TS), ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) for MRSA at 53 °C in digested sludge .........................38 Figure 2-7. ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) for MRSA at 53 °C in digested sludge for an extended experiment length of 60 days ................................................................................................................................39 Figure 2-8. Comparison of ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) in Day 60 samples for MRSA at 53 °C in digested sludge for an extended experiment length of 60 days .................................................................40 Figure 2-9. Plate counts (CFU/ml PBS), ARG (mecA) and MRSA-specific gene (nuc) (gene copy numbers (gcn/ml PBS) for MRSA at 70 °C in PBS ......................................41 Figure 2-10. Plate counts (CFU/g TS), ARGs (mecA, tet(G)) and MRSA-specific gene (nuc) (gene copy numbers (gcn/g TS) for MRSA at 70 °C in digested sludge ...............42 Figure 2-11. Plate counts (CFU/ml PBS), ARGs (tet(A), sul2, tet(G)) and E. coli-specific gene (gadA/B) for E. coli at 37 °C in PBS .....................................................................43 Figure 2-12. Plate counts (CFU/g TS), ARGs (tet(A), sul2, tet(G)) and E. coli-specific gene (gadA/B) for E. coli at 37 °C in digested sludge ...................................................44 Figure 2-13. Plate counts (CFU/ml PBS), ARGs (tet(A), sul2, tet(G)) and E. coli-specific gene (gadA/B) for E. coli at 53 °C at 53 °C in PBS .......................................................45 Figure 2-14. Plate counts (CFU/g TS), ARGs (tet(A), sul2, tet(G)) and E. coli-specific gene (gadA/B) for E. coli at 53 °C in digested sludge ...................................................46 Figure 2-15. ARGs (tet(A), sul2, tet(G)) and E. coli-specific gene (gadA/B) (gene copy numbers (gcn/g TS) for E. coli at 53 °C in digested sludge for an extended experiment length of 60 days ........................................................................................................................47 vii Figure 3-1. Plate count (CFU/ml) and ARG and 16S rRNA (gcn/ml) for tet(G) associated with tetracycline-resistant isolate M1-1 and tet(W) in the background community at 37 °C in digested sludge .......................................................................................................66 Figure 3-2. Normalized ARG ratios (ARG gcn / 16S rRNA) for tet(G) associated with tetracycline-resistant isolate M1-1 and tet(W) in the background community at 37 °C in digested sludge .......................................................................................................67 Figure 3-3. Plate count (CFU/ml) and ARG and 16S rRNA (gcn/ml) for tet(W) associated with tetracycline-resistant isolate T10 and tet(G) in the background community at 53 °C in digested sludge .......................................................................................................68 Figure 3-4. Normalized ARG ratios (ARG gcn / 16S rRNA) for tet(W) associated with tetracycline-resistant isolate T10 and tet(G) in the background community at 53 °C in digested sludge .......................................................................................................69 Figure 3-5. ARG concentrations including intI1 (Figure 3-5a), sul1 (Figure 3-5b), tet(O) (Figure 3-5c), and tet(W) (Figure 3-5d) in raw sludge, mesophilic digester effluent, and thermophilic digester effluent during nine months of monitoring of semi-continuously fed lab-scale digesters ..................................................................................................70 Figure 4-1. Comparison of volatile solids reduction (%, VSR), pH, gas volume, and methane composition during all dosing levels .....................................................................92 Figure 4-2. ARG and intI1 ratios in the raw feed sludge (Feed), test digesters (Ag NP, Ionic silver, and SMX), thermophilic control digester (Thermo Control), and mesophilic control digester (Meso Control) .............................................................................93 Figure 4-3. Quantities of ARGs and intI1and 16S rRNA gene copy numbers (number per µl sludge) in the raw feed sludge (Feed), test digesters (Ag NP, Ionic silver, and SMX), thermophilic control digester (Thermo Control), and mesophilic control digester (Meso Control) ..................................................................................................................94 Figure 4-4. sul1 and intI1 gene copy numbers and ARG ratios during the Level 2 steady state sampling period ......................................................................................................95 Figures 5-1a and 5-1b. For biosolids stored at 4 °C, ARG (intI1 and sul1) gene copy numbers normalized to TS (Figure 1a), VS (Figure 1a), and 16S rRNA (Figure 1b) expressed in units of gene copy numbers per kg solids (gcn/kg) .............................................105 Figures 5-2a and 5-2b. For biosolids stored at 10 °C, ARG (intI1 and sul1) gene copy numbers normalized to TS (Figure 2a), VS (Figure 2a),, and 16S rRNA (Figure 2b) expressed in units of gene copy numbers per kg solids (gcn/kg) .............................................106 Figures 5-3a and 5-3b. For biosolids stored at 20 °C, ARG (intI1 and sul1) gene copy numbers normalized to TS (Figure 3a), VS (Figure 3a), and 16S rRNA (Figure 3b) expressed in units of gene copy numbers per kg solids (gcn/kg) .............................................107 Figures S5-1a and S5-1b. TS and VS (mg/L, Figure S1a) and intI1, sul1, and 16S rRNA gene copy numbers per milliliter digested sludge (gcn/µl, Figure S1b) for experimental triplicates of biosolids stored at 4 °C ...................................................................110 viii Figures S5-2a and S5-2b. TS and VS (mg/L, Figure S2a) and intI1, sul1, and 16S rRNA gene copy numbers per milliliter digested sludge (gcn/µl, Figure S2b) for experimental triplicates of biosolids stored at 10 °C .................................................................111 Figures S5-3a and S5-3b. TS and VS (mg/L, Figure S1a) and intI1, sul1, and 16S rRNA gene copy numbers per milliliter digested sludge (gcn/µl, Figure S3b) for experimental triplicates of biosolids stored at 20 °C .................................................................112 ix ATTRIBUTION Each coauthor is duly credited for his or her contribution to this work, both in their sharing of ideas and technical expertise. Research assistants are also noted for their contribution to experiment sample collection and analysis. Amy Pruden, PhD. Professor of Civil and Environmental Engineering Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Coauthor of chapters 2, 3, 4, and 5 William R. Knocke, Ph.D., P.E. W.C. English Professor of Civil and Environmental Engineering Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Coauthor of chapters 2, 3, 4, and 5 John T. Novak, Ph.D., P.E. Nick Prillaman Professor of Environmental Engineering Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Coauthor of chapters 2,3,4, and 5 Peter J. Vikesland, Ph.D. Professor of Civil and Environmental Engineering Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Coauthor of chapter 5 Matthew Hull, Ph.D. Program Manager, Nano-Bio Interface and Nanoscale Science and Engineering, Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Coauthor of chapter 2 Sudhir N. Murthy, Ph.D., P.E. Manager of Process Development and Optimization Blue Plains Advanced Wastewater Treatment Plant, District of Columbia Water and Sewer Authority. Washington D.C. 20052 Coauthor of chapter 2 Charles B. Bott, Ph.D., P.E. Research and Development Manager Hampton Roads Sanitation District PO Box 5911, Virginia Beach, VA 23471-0911 Coauthor of chapter 2 Matthew J. Higgins, , Ph.D., P.E. Claire W. Carlson Chair in Environmental Engineering Department of Civil and Environmental Engineering Bucknell University, Lewisburg, PA 17837 Coauthor of chapter 2 x