Effect of Different Treatment Technologies on the Fate of Antibiotic

Nov 17, 2017 - University of Minnesota, Department of Civil, Environmental, and Geo- Engineering, 500 Pillsbury Drive SE, Minneapolis, MN 55455. ...
0 downloads 15 Views 965KB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

The Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil Tucker Burch, Michael J. Sadowsky, and Timothy M. LaPara Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04760 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Environmental Science & Technology

The Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil Tucker R. Burch,1 Michael J. Sadowsky,2, 3, 4 and Timothy M. LaPara1,2* 1

University of Minnesota Department of Civil, Environmental, and Geo- Engineering Minneapolis, MN 55455 2

University of Minnesota Biotechnology Institute St. Paul, MN 55108

3

University of Minnesota Department of Soil, Water, and Climate St. Paul, MN 55108 4

University of Minnesota Department of Plant and Microbial Biology St. Paul, MN 55108



Current affiliation: USDA-ARS, Environmentally Integrated Dairy Management Research Unit, Marshfield, WI 54449 *

Correspondence: Timothy M. LaPara, University of Minnesota, Department of Civil, Environmental, and Geo- Engineering, 500 Pillsbury Drive SE, Minneapolis, MN 55455. Tel: (612) 624-6028. Fax: (612) 626-7750. Email: [email protected] Word Count: 3,092 + two figures (300+300) and three tables (300+300+600) = 5,092

ACS Paragon Plus Environment

Environmental Science & Technology

39

ABSTRACT

40

Residual wastewater solids are a significant reservoir of antibiotic resistance genes

41

(ARGs). While treatment technologies can reduce ARG levels in residual wastewater solids, the

42

effects of these technologies on ARGs in soil during subsequent land-application are unknown.

43

In this study we investigated the use of numerous treatment technologies (air drying, aerobic

44

digestion, mesophilic anaerobic digestion, thermophilic anaerobic digestion, pasteurization, and

45

alkaline stabilization) on the fate of ARGs and class 1 integrons in wastewater solids-amended

46

soil microcosms. Six ARGs (erm(B), qnrA, sul1, tet(A), tet(W), and tet(X)), the integrase gene

47

of class 1 integrons (intI1), and 16S rRNA genes were quantified using quantitative PCR. The

48

quantities of ARGs and intI1 decreased in all microcosms, but thermophilic anaerobic digestion,

49

alkaline stabilization, and pasteurization led to the most extensive decay of ARGs and intI1,

50

often to levels similar to the control microcosms to which no wastewater solids had been applied.

51

In contrast, the rates by which ARGs and intI1 declined using the other treatment technologies

52

were generally similar, typically varying by less than 2-fold. These results demonstrate that

53

wastewater solids treatment technologies can be used to decrease the persistence of ARGs and

54

intI1 during their subsequent application to soil.

1

ACS Paragon Plus Environment

Page 2 of 28

Page 3 of 28

55

Environmental Science & Technology

TOC ART

56 57 58

2

ACS Paragon Plus Environment

Environmental Science & Technology

59 60

INTRODUCTION Bacterial resistance to antibiotics is a major public health dilemma with serious health

61

and financial consequences.1,2 Numerous strategies are being pursued to mitigate the effects of

62

antibiotic resistance, including the development of new or alternative antibiotics, the

63

development of alternatives to antibiotics, and the reduction of antibiotic use for purposes other

64

than treating diseases in humans.3-8 An alternative and complementary strategy consists of

65

identifying and mitigating antibiotic resistance genes (ARGs) as environmental contaminants.9

66

This has led to the identification of numerous environmental reservoirs of ARGs, including

67

surface waters, aquaculture facilities, and agricultural waste.9-23 One of the largest reservoirs,

68

however, appears to be untreated municipal wastewater and the residual municipal wastewater

69

solids produced from the wastewater treatment process.9, 14, 24-34 The treatment of residual

70

wastewater solids, therefore, may be an excellent opportunity to mitigate the release of ARGs to

71

the environment.

72

Previous research has demonstrated the potential for existing treatment technologies to

73

remove ARGs from residual solids. These technologies, referred to as Processes to Significantly

74

Reduce Pathogens (PSRPs) and Processes to Further Reduce Pathogens (PFRPs),36 are currently

75

designed to reduce the water, organic carbon, and pathogen content of untreated residual

76

municipal wastewater solids.37 However, PSRPs and PFRPs have also been demonstrated to

77

remove ARGs from untreated residual municipal wastewater solids,38-41 both by reducing the

78

overall number of microorganisms in the residual solids and by creating environmental

79

conditions that fail to select for particular ARGs based on their ecological niches.41-42 Aerobic

80

digestion, air-drying beds, and anaerobic digestion have all been demonstrated to remove ARGs

81

from residual municipal wastewater solids to varying degrees. Moreover, ARG removal can be

3

ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28

Environmental Science & Technology

82

optimized by adding pretreatment steps, increasing treatment temperatures, and inducing feast-

83

famine (i.e. batch) cycles.38-42

84

The degree to which all of these technologies affect the fate of ARGs in soil following

85

land-application of treated residual solids, however, is unknown. This is a significant gap in the

86

body of knowledge, because approximately 50% of all treated residual municipal wastewater

87

solids are land-applied in the United States.43 Furthermore, previous work has indicated that

88

ARGs originating in anaerobically-digested (35°C) residual solids can persist for months at

89

relatively high concentrations in soil following land-application.44 This leads to the potential

90

opportunity for off-site transport of, and ultimately human exposure to, ARGs with stormwater

91

or airborne particulate matter.45-46 Many current PSRP and PFRP technologies are widely used

92

in the U.S.43 or could be retrofitted to existing infrastructure with relative ease. Thus, their

93

implementation for controlling the quantities of ARGs circulating in the environment could

94

represent a tractable management strategy for preventing antibiotic resistant infections.

95

The objective of this work was to determine how different technologies used to treat

96

residual wastewater solids affected the persistence of ARGs and class 1 integrons in soil

97

following land-application of treated residual solids. The treatment technologies examined

98

included aerobic digestion at 17°C, air drying, alkaline stabilization, pasteurization, and

99

anaerobic digestion at 38°C, 55°C, 63°C and 69°C. In addition, untreated residual municipal

100

wastewater solids were examined to represent a “worst-case scenario” control for the application

101

of ARGs to soil. Residual solids from each of the nine sources were added to soil microcosms,

102

and the quantities of several ARGs and the integrase gene of class 1 integrons were determined

103

in each microcosm via quantitative PCR over a six-month time series.

4

ACS Paragon Plus Environment

Environmental Science & Technology

104 105

MATERIALS AND METHODS Experimental Design. Soil microcosms were constructed as described previously.44

106

Briefly, triplicate microcosms were used for each experiment were comprised of a mixture of

107

200 g of agricultural soil (Waukegan silt loam; fine-silty, mixed, mesic Typic Hapludoll; initial

108

moisture content of 12.1% ± 0.3%) thoroughly mixed with 8 g (wet mass) of residual wastewater

109

solids. No manure or wastewater solids had been previously applied to the soil. This mass

110

loading rate of residual wastewater solids to soil is typical of common practices.37 All

111

microcosms were stored at room temperature (approximately 20°C). Evaporative water losses

112

for all microcosms were estimated by weighing the microcosms at approximately monthly

113

intervals and assuming that all mass loss was due to evaporation of water.

114

Untreated wastewater solids were obtained from a municipal wastewater treatment

115

facility in south-central Minnesota. These solids were then treated by various laboratory-scale

116

simulations of full-scale processes commonly used to treat wastewater solids. Air-dried solids,41

117

aerobically-digested solids 40 and anaerobically digested solids42 were collected from the

118

laboratory-scale treatment units described previously. To produce alkaline stabilized residual

119

solids, the pH of untreated residual solids was increased to above 12 for a period of two hours

120

using sodium hydroxide. The pH was then neutralized using hydrochloric acid. To produce

121

pasteurized residual solids, the temperature of untreated residual solids was increased to more

122

than 70°C for a period of 30 minutes.

123

Sample Collection and Genomic DNA Extraction. Microcosms were mixed prior to

124

each sample collection event and then triplicate samples of ~0.5 g were collected from each the

125

triplicate soil microcosms. Samples were stored at -20 °C until DNA was extracted and purified

5

ACS Paragon Plus Environment

Page 6 of 28

Page 7 of 28

Environmental Science & Technology

126

using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) and a BIO 101 Thermo

127

Savant FastPrep FP120 Cell Disruptor (Qbiogene, Inc., Carlsbad, CA).

128

Real-time PCR. The forward and reverse primer sequences, expected amplicon sizes,

129

and primer annealing temperatures for all gene targets are provided in the Supporting

130

Information (Table S1). The genes targeted in this study were selected based on prior studies that

131

demonstrated each of these genes is easily quantifiable in untreated wastewater solids.38, 40-42, 44

132

The number of standards, slope, intercept, amplification efficiency, r2, and quantification limit

133

for each standard curve are also provided in the Supporting Information (Table S2). Of the 58

134

assays performed, 26 (45%) had amplification efficiencies of 100% ± 10% (maximum deviation

135

from 100%) with r2 ≥ 0.99 for a minimum of five points on each standard curve. The absolute

136

values by which amplification efficiencies for the remaining 32 assays deviated from 100% were

137

between 11% and 27% (median of 15%) with r2 ≥ 0.98 for a minimum of five points on each

138

standard curve. No template controls were performed during all qPCR assays. The amplification

139

efficiencies of experimental samples were visually inspected to ensure that their amplification

140

efficiency matched that of the standards.

141

Data Analysis. Prior experience with qPCR analysis of ARGs suggested that the data

142

are best described by a log10-normal distribution.41-42, 44 In this current study, therefore, the data

143

were log10-transformed prior to statistical analysis. Tukey’s honest significance difference test

144

was performed using JMP Pro. Ver. 12.0.1 (SAS Institute, Inc.; Cary, NC).

145 146

ARG data from the soil microcosms were fit to a modified Collins-Selleck model47-48 using the following equation:  

  =   −   

6

ACS Paragon Plus Environment

Environmental Science & Technology

147

In this equation, N is the number of ARG copies at time t, N0 is the initial quantity of

148

ARG, ΛCS is the specific lethality coefficient, and b is the lag coefficient. This modified Collins-

149

Selleck model has been previously used to fit ARG decay data in anaerobic digestors42 and in

150

soil microcosms.49 ARG data from the soil microcosms were also fit to a first-order model, as

151

described previously,44 except that the initial time point was excluded from analysis.

152 153 154

RESULTS Untreated wastewater solids were collected from a full-scale municipal wastewater

155

treatment facility and processed using numerous bench-scale simulations of full-scale

156

technologies used to treat wastewater solids (air drying, aerobic digestion, mesophilic anaerobic

157

digestion, thermophilic anaerobic digestion, pasteurization, and alkaline stabilization). These

158

approaches generated treated wastewater solids that contained relatively similar quantities of

159

bacterial biomass (i.e., within one log10 unit), but markedly different levels of ARGs and class 1

160

integrons (Table 1).

161

Negative control soil microcosms (i.e., microcosms to which no wastewater solids were

162

applied) were described previously.44 Briefly, sul1 and intI1 were detected in about 50% of the

163

samples in the negative control microcosms at concentrations near the detection limit (~103 gene

164

copies per gram of wet soil), whereas erm(B) and tet(X) were detected less frequently. In

165

contrast, tet(A) and tet(W) were never detected in the negative control soil microcosms.

166

The quantities of bacterial biomass, measured as 16S rRNA gene copy number, generally

167

followed similar trends for all experimental treatments. The quantities of 16S rRNA genes were

168

statistically unchanged (P > 0.05) throughout the experiment for most experimental treatments,

169

ranging in concentration from 1.5 1010 gene copies per g of soil (alkaline stabilization) to 9.3

7

ACS Paragon Plus Environment

Page 8 of 28

Page 9 of 28

Environmental Science & Technology

170

1010 gene copies per g of soil (anaerobic digestion at 38°C). In the soil microcosms receiving

171

untreated wastewater solids or wastewater solids treated by aerobic digestion or pasteurization,

172

the quantities of 16S rRNA genes declined (P ≤ 0.05) at relatively slow rates, with half-lives

173

varying between 120 day (aerobic digestion) and 230 days (wastewater solids without treatment).

174

Three common trends were observed in the quantities of ARGs and intI1 after treated

175

wastewater solids had been applied (Figs. 1-2; Figs. S1-S5): (1) a pattern in which the quantities

176

of ARGs rapidly declined initially (i.e., < 30 days), but then subsequently declined at a much

177

slower rate. This trend could be be modeled using a modified Collins-Selleck model or using a

178

first-order model that neglected the initial time point.; (2) the quantities of ARGs and intI1

179

followed a first-order model throughout the experiment; and (3) the quantities of ARGs (intI1 did

180

not exhibit this trend) were initially detectable but then declined such that they could no longer

181

be detected. This last trend was not fit to any kinetic model.

182

The rates at which different ARGs and intI1 decayed in soils exhibited a myriad of

183

effects with respect to the technology used to treat the wastewater solids prior to soil application,

184

independent of which regression model was applied (Table 2). For many of the combinations of

185

genes and treatment technologies, decay coefficients could not be computed because gene

186

quantities declined below the detection limits of the assays. Presumably, these treatment

187

technologies either significantly reduced the quantities of these genes such that the quantities

188

were at or below the detection limits at the initiation of the soil microcosm experiments or these

189

genes decayed very rapidly such that decay coefficients could not be computed. This inability to

190

regress decay coefficients occurred most commonly with wastewater solids treated by

191

thermophilic anaerobic digestion, which achieved substantial removal of all of these genes prior

192

to application to soils.42 In contrast, pasteurization and alkaline stabilization did not achieve

8

ACS Paragon Plus Environment

Environmental Science & Technology

193

substantial removal of ARGs (Fig. S6) even though it is likely that both of these technologies

194

were very effective at killing the bacteria in the wastewater solids that harbored these genes;

195

thus, the cells almost certainly died, but their DNA remained intact and susceptible to

196

amplification via PCR. In these cases, the inability to compute a decay rate was likely due to

197

rapid decay rather than a low initial quantity of ARGs in the soils. Decay coefficients could be

198

computed for intI1 and sul1, regardless of the wastewater solids treatment technology. With

199

both of these genes, the decay coefficients were relatively similar, varying by less than an order

200

of magnitude, independent of the technology used to treat wastewater solids.

201

A substantial variation was observed in the quantities of ARGs after the soil microcosms

202

had been incubated for six months (Table 3). In many cases, the quantities of ARGs in the soils

203

were below the detection limits, similar to the control experiments. This result was most

204

common for ARGs following thermophilic anaerobic digestion. Only two genes (intI1 and sul1)

205

were quantifiable in all microcosms, independent of the treatment technology. Thermophilic

206

anaerobic digestion (55°C and 69°C), pasteurization, and alkaline stabilization led to the

207

statistically lowest quantities of intI1. Similarly, the same upstream treatment technologies led

208

to the statistically lowest quantities of sul1.

209 210

DISCUSSION

211

There is a growing body of literature that has identified numerous reservoirs of antibiotic

212

resistant bacteria and ARGs in the environment.50-54 Untreated municipal wastewater appears to

213

be a particularly large reservoir of ARGs,30, 55 of which more than 90% leave municipal

214

wastewater treatment facilities with the residual wastewater solids.30 Because wastewater solids

215

are commonly applied to agricultural soils, it is critically important to understand the fate of

9

ACS Paragon Plus Environment

Page 10 of 28

Page 11 of 28

Environmental Science & Technology

216

ARGs in soils after land-application. While previous studies have explored the fate of ARGs

217

following the application of wastewater solids to soil,44, 56 the present study provides new

218

knowledge by delineating the role of the various treatment technologies on the rate and extent by

219

which ARGs decay in soil. This research demonstrates that treatment technologies already

220

known to aggressively inactivate pathogens in wastewater solids, such as thermophilic anaerobic

221

digestion, alkaline stabilization, and pasteurization (most of these are PFRPs), lead to more

222

extensive decay of ARGs than less aggressive treatment technologies, such as aerobic digestion,

223

mesophilic anaerobic digestion, and air drying.

224

The various technologies used to treat wastewater solids had only a marginal effect on the

225

rates (usually less than 2-fold) of decay of ARGs and intI1 after residual wastewater solids were

226

applied to soil. Similarly, pasteurization and alkaline stabilization often led to comparatively

227

rapid decay rates when treated wastewater solids were applied to soil, albeit these rates are likely

228

an artifact of qPCR. We failed to observe substantial decay of ARGs during the pasteurization

229

and alkaline stabilization of wastewater solids (Fig. S6), even though both of these technologies

230

are known to aggressively inactivate bacteria.57 We assumed that pasteurization and alkaline

231

stabilization processes successfully inactivated most bacteria in the wastewater solids during the

232

relatively short, but lethal, incubations at high temperature and pH, respectively. Their DNA,

233

however, remained intact, thus leading to artificially high quantifications of ARGs and intI1 at

234

the initiation of our soil microcosm experiments. The quantities of these genes then decayed at

235

comparatively rapid rates in soil because they were harbored by dead organisms. In contrast,

236

thermophilic anaerobic digestion was able to achieve substantial ARG and intI1 removal prior to

237

soil application, such that the subsequent decay of ARGs and intI1 were slower because they

238

were harbored by viable organisms.

10

ACS Paragon Plus Environment

Environmental Science & Technology

239

The various technologies used to treat wastewater solids had a substantial effect on the

240

extent of decay of ARGs and intI1 after residual wastewater solids were applied to soil. The

241

treatment technologies that are known to aggressively inactivate human pathogens (thermophilic

242

anaerobic digestion, pasteurization, and alkaline stabilization) usually resulted in either ARG

243

levels that were below the detection limit of our qPCR assays (i.e., similar to our negative

244

control microcosms) or were statistically the lowest levels of our experimental comparisons

245

(albeit higher than our negative control microcosms). This research, therefore, makes a seminal

246

contribution to new knowledge by identifying a potentially sustainable approach to managing

247

ARGs and intI1 genes during wastewater treatment. These results are substantially different than

248

what was previously reported for the application of untreated animal manure in which ARG

249

levels in soils did not return to pre-manure-application levels within six months.49, 58 Thus, our

250

research also suggests that the treatment of animal manure prior to application to soil could be

251

beneficial for limiting the spread of antibiotic resistance from animal feeding operations.

252

The present study demonstrated that the fate of ARGs and intI1 in wastewater solids

253

applied to soils can be kinetically-modeled, as has been observed previously when animal

254

manure was applied to soil.49 The ability to model the decay of ARGs is of importance because

255

it allows different studies and experiments to be compared and because it allows for predictions

256

on the fate of ARGs in future applications. Although the utility of kinetic modeling in

257

understanding and predicting the fate of ARGs is clear, relatively few studies thus far have

258

performed kinetic modeling.

259

The kinetic modeling used herein suggests that there are multiple mechanisms by which

260

ARGs decay after wastewater solids are applied to soil. In many of our experiments, ARGs

261

initially declined rapidly but then slowed substantially. In this study, we modeled these

11

ACS Paragon Plus Environment

Page 12 of 28

Page 13 of 28

Environmental Science & Technology

262

phenomena with a modified Collins-Selleck model, similar to prior experiments on the fate of

263

ARGs during thermophilic anaerobic digestion42 and when animal manure was applied to soils.49

264

Alternatively, the data could likely be modeled using two first-order models, as previously used

265

to describe aerobic digestion of wastewater solids,40 although this approach was not generally

266

feasible in this study due to the lack of data during the initial period during which ARG decay

267

was rapid. We hypothesize that the initial rapid decay of ARGs occurs because inactivated cells

268

and bacteria incapable of living in soil perish and release their DNA, which is then rapidly turned

269

over via normal biogeochemical cycling. Additional research is needed to test this hypothesis as

270

well as to understand the mechanisms by which ARGs appear to slowly decay in soil after this

271

initial period of rapid decay.

272

This study identified approaches to better remove class 1 integrons from wastewater

273

solids. Integrons are believed to be particularly pertinent for the spread of antibiotic resistance

274

because they enable both horizontal gene transfer and the accumulation of multiple ARGs in an

275

individual bacterium.59-60 In fact, integrons have been shown to contain multiple ARGs in

276

environmental samples.61-65 Similar to the ARGs, intI1 genes were best removed by treatment

277

technologies known to aggressively inactivate human pathogens (thermophilic anaerobic

278

digestion, pasteurization, and alkaline stabilization). Curiously, the application of untreated

279

wastewater solids to soils resulted in a greater extent of intI1 decay than if air drying, aerobic

280

digestion, or mesophilic anaerobic digestion was used to treat the wastewater solids prior to soil-

281

application.

282

Although the experiments described here uncovered important information regarding the

283

fate of ARGs and class 1 integrons when wastewater solids are applied to soil, our study also

284

suffered from several limitations due to our experimental design. The qPCR assays used in this

12

ACS Paragon Plus Environment

Environmental Science & Technology

285

work cannot distinguish between genes in viable cells from extracellular DNA or from DNA of

286

dead but intact bacteria. This is pertinent because a substantial fraction of ARGs can be

287

extracellular66-67 or in inactivated cells.57 Another limitation is that the microbial hosts of the

288

ARGs and intI1 genes remain unknown. This is pertinent because ARGs are more worrisome in

289

pathogens than in bacteria benign to humans. Similarly, ARGs have been found in bacteria

290

lacking the capability of expressing these genes.68 Our study was also limited because the soil

291

microcosms used were kept under the controlled conditions of a laboratory rather than being

292

exposed to highly variable field conditions, which include direct sunlight, natural fluctuations in

293

moisture content and temperature, and the presence of other organisms. Finally, the work

294

presented in this study investigates only a small fraction of known ARGs. Other researchers

295

have quantified ARGs by shotgun metagenomics,69-70 although the sensitivity of shotgun

296

metagenomics is poor compared to qPCR. Similarly, other researchers have developed assays to

297

simultaneously track more than 250 ARGs and mobile genetic elements by PCR,23, 56 although

298

we question the validity of these PCR arrays to provide quantitative information because they

299

typically do not use external standards for more than a single gene.

300 301 302

SUPPORTING INFORMATION The Supporting Information contains data regarding the qPCR primers, conditions, and

303

performance (Tables S1-S2), a table of the extent of ARG and intI1 removal in the soil

304

microcosms (Table S3), the quantities of other ARGs and intI1 in all experimental microcosms

305

(Figures S1-S5), and the quantities of 16S rRNA genes, intI1, and tet(X) during pasteurization

306

and alkaline stabilization experiments (Figure S6). This information is available free of charge

307

via the Internet at http://pubs.acs.org/.

13

ACS Paragon Plus Environment

Page 14 of 28

Page 15 of 28

Environmental Science & Technology

308 309 310

ACKNOWLEDGEMENTS This work was supported by National Science Foundation grant 0967176.

311 312

REFERENCES

313 314 315

1. Antibiotic Resistance Threats in the United States, 2013. Centers for Disease Control and Prevention, U.S. Department of Health and Human Services: Washington D.C., 2013; http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf

316 317 318 319 320

2. Roberts, R. R.; Hota, B.; Ahmad, I.; Scott, R. D.; Foster, S. D.; Abbasi, F.; Schabowski, S.; Kampe, L. M.; Ciavarella, G. G.; Supino, M.; Naples, J.; Cordell, R.; Levy, S. B.; Weinstein, R. A. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clin. Infect. Dis. 2009, 49, 1175–1184.

321 322

3. Jabes, D. The antibiotic R&D pipeline: an update. Curr. Opin. Microbiol. 2011, 14, 564– 569.

323 324

4. Alekshun, M. N.; Levy, S. B. Targeting virulence to prevent infection: to kill or not to kill? Drug Discovery Today: Ther. Strategies 2004, 1, 483–489.

325 326 327

5. Arnold, S.; Gassner, B.; Giger, T.; Zwahlen, R. Banning antimicrobial growth promoters in feedstuffs does not result in increased therapeutic use of antibiotics in medicated feed in pig farming. Pharmacoepidemiol. Drug Saf. 2004, 13, 323– 331.

328 329 330

6. Edgar, R.; Friedman, N.; Molshanski-Mor, S.; Qimron, U. Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. Appl. Environ. Microbiol. 2012, 78, 744–751.

331 332

7. Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. How antibiotics kill bacteria: from targets to networks. Nat. Rev. Microbiol. 2010, 8, 423–435.

333 334

8. Merril, C. R.; Scholl, D.; Adhya, S. L. The prospect for bacteriophage therapy in Western medicine. Nat. Rev. Drug Discovery 2003, 2, 489–497.

335 336 337

9. Pruden, A.; Pei, R.; Storteboom, H.; Carlson, K. H. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environ. Sci. Technol. 2006, 40, 7445–7450.

338 339

10. Chee-Sanford, J.C.; Aminov, R. I.; Krapac, I. J.; Garrigues-Jeanjean, N.; Mackie, R. I. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater

14

ACS Paragon Plus Environment

Environmental Science & Technology

340 341

underlying two swine production facilities. Appl. Environ. Microbiol. 2001, 67, 1494– 1502.

342 343 344

11. Chen, J.; Min, J.; Qiu, Z.-G.; Guo, C.; Chen, Z.-L.; Shen, Z.-Q.; Wang, X.-W.; Li, J.-W. A survey of drug resistance bla genes originating from synthetic plasmid vectors in six Chinese rivers. Environ. Sci. Technol. 2012, 46, 13448-13454.

345 346 347 348

12. Cummings, D. E.; Archer, K. F.; Arriola, D. J.; Baker, P. A.; Faucett, K. G.; Laroya, J. B.; Pfeil, K. L.; Ryan, C. R.; Ryan, K. R. U.; Zuill, D. E. Broad dissemination of plasmid-mediated quinolone resistance genes in sediments of two urban coastal wetlands. 2011, Environ. Sci. Technol., 45, 447-454.

349 350 351

13. Graham, D. W.; Olivares-Rieumont, S.; Knapp, C. W.; Lima, L.; Werner, D.; Bowen, E. Antibiotic resistance gene abundances associated with waste discharges to the Almendares River near Havana, Cuba. Environ. Sci. Technol. 2011, 45, 418-424.

352 353 354

14. LaPara, T. M.; Burch, T. R.; McNamara, P. J.; Tan, D. T.; Yan, M.; Eichmiller, J. J. Tertiary-treated municipal wastewater is a significant point source of antibiotic resistance genes into Duluth-Superior Harbor. Environ. Sci. Technol. 2011, 45, 9543–9549.

355 356 357

15. Li, J.; Shao, B.; Shen, J.; Wang, S.; Wu, Y. Occurrence of chloramphenicol-resistance genes as environmental pollutants from swine feedlots. Environ. Sci. Technol. 2013, 47, 2892-2897.

358 359 360

16. Peak, N.; Knapp, C. W.; Yang, R. K.; Hanfelt, M. M.; Smith, M. S.; Aga, D. S.; Graham, D. W. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ. Microbiol. 2007, 9, 143-151.

361 362 363

17. Pei, R.; Kim, S. C.; Carlson, K. H.; Pruden, A. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res. 2006, 40, 2427–2435.

364 365

18. Pruden, A.; Arabi, M.; Storteboom, H. N. Correlation between upstream human activities and riverine antibiotic resistance genes. Environ. Sci. Technol. 2012, 46, 11541-11549.

366 367 368

19. Schwartz, T.; Kohnen, W.; Jansen, B.; Obst, U. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol Ecol. 2003, 43, 325-335.

369 370 371

20. Seyfried, E. E.; Newton, R. J.; Ribert, K. F.; Pedersen, J. A.; McMahon, K. D. Occurrence of tetracycline resistance genes in aquaculture facilities with varying use of oxytetracycline. Microb. Ecol. 2010, 59, 799-807.

372 373 374

21. Storteboom, H.; Arabi, M.; Davis, J. G; Crimi, B.; Pruden, A. Tracking antibiotic resistance genes in the South Platte River basin using molecular signatures of urban, agricultural, and pristine sources. Environ. Sci. Technol. 2010, 44, 7397-7404.

15

ACS Paragon Plus Environment

Page 16 of 28

Page 17 of 28

Environmental Science & Technology

375 376 377

22. Tamminen, M.; Karkman, A.; Löhmus, A.; Muziasari, W. I.; Takasu, H.; Wada, S.; Suzuki, S.; Virta, M. Tetracycline resistance genes persist at aquaculture farms in the absence of selection pressure. Environ. Sci. Technol. 2011, 45, 386–391.

378 379 380

23. Zhu, Y.-G.; Johnson, T. A.; Su, J. Q.; Qiao, M.; Guo, G. X.; Stedtfeld, R. D.; Hashsham, S. A.; Tiedje, J. M. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Nat. Acad. Sci. USA 2013, 110, 3435-3440.

381 382

24. Auerbach, E. A.; Seyfried, E. E.; McMahon, K. D. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res. 2007, 41, 1143-1151.

383 384 385

25. Börjesson, S.; Melin, S.; Matussek, A.; Lindgren, P.-E. A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant. Water Res. 2009, 43, 925–932.

386 387 388 389

26. Börjesson, S.; Dienues, O.; Jarnheimer, P.-A.; Olsen, B.; Matussek, A.; Lindgren, P.-E. Quantification of genes encoding resistance to aminoglycosides, β-lactams and tetracyclines in wastewater environments by real-time PCR. Int. J. Environ. Health Res. 2009, 19, 219–230.

390 391 392

27. Ghosh, S., Ramsden, S. J.; LaPara, T. M. The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants. Appl. Microbiol. Biotechnol. 2009, 84, 791-796.

393 394 395

28. Kim, S.; Park, H.; Chandran, K. Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: a mathematical modeling approach. Chemosphere 2010, 78, 1071–1077.

396 397 398

29. Lachmayr, K. L.; Kerkhof, L. J.; Dirienzo, A. G.; Cavanaugh, C. M.; Ford, T. E. Quantifying nonspecific TEM β-lactamase (blaTEM) genes in a wastewater stream. Appl. Environ. Microbiol. 2009, 75, 203–211.

399 400 401

30. Munir, M.; Wong, K.; Xagoraraki, I. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan. Water Res. 2011, 45, 681–693.

402 403 404

31. Uyaguari, M. I.; Fichot, E. B.; Scott, G. I.; Norman, R. S. Characterization and quantitation of a novel β-lactamase gene within a wastewater treatment facility and surrounding coastal ecosystem. Appl. Environ. Microbiol. 2011, 77, 8226–8233.

405 406 407

32. Volkmann, H.; Schwartz, T.; Bischoff, P.; Kirchen, S.; Obst, U. Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan). J. Microbiol. Methods 2004, 56, 277–286.

408 409 410

33. Zhang, X.-X.; Zhang, T. Occurrence, abundance, and diversity of tetracycline resistance genes in 15 sewage treatment plants across China and other global locations. Environ. Sci. Technol. 2011, 45, 2598–2604.

16

ACS Paragon Plus Environment

Environmental Science & Technology

411 412 413

34. Zhang, T.; Zhang, M.; Zhang, X.; Fang, H. H. Tetracycline resistance genes and tetracycline resistant Enterobacteriaceae in activated sludge of sewage treatment plants. Environ. Sci. Technol. 2009, 43, 3455–3460.

414 415 416

35. Zhang, X.-X.; Zhang, T.; Zhang, M.; Fang, H. H. P.; Cheng, S.-P. Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants. Appl. Microbiol. Biotechnol. 2009, 82, 1169–1177.

417 418

36. Standards for the Use or Disposal of Sewage Sludge. Code of Federal Regulations, Part 503, Title 40, 2007.

419 420

37. Tchobanoglous, G.; Burton, F. L.; Stensel, H. D. Wastewater Engineering: Treatment and Reuse; 4th ed.; McGraw-Hill: Boston, MA, 2003.

421 422 423 424

38. Diehl, D. L.; LaPara, T. M. Effect of temperature on the fate of genes encoding tetracycline resistance and the integrase of class 1 integrons within anaerobic and aerobic digesters treating municipal wastewater solids. Environ. Sci. Technol. 2010, 44, 9128– 9133.

425 426 427

39. Ma, Y.; Wilson, C. A.; Novak, J. T.; Riffat, R.; Aynur, S.; Murthy, S.; Pruden, A. Effect of various sludge digestion conditions on sulfonamide, macrolide, and tetracycline resistance genes and class I integrons. Environ. Sci. Technol. 2011, 45, 7855–7861.

428 429 430

40. Burch, T. R.; Sadowsky, M. J.; LaPara, T. M. Aerobic digestion reduces the quantity of antibiotic resistance genes in residual municipal wastewater solids. Front. Microbiol. 2013, 4(17), 1-9; DOI: 10.3389/fmicb.2013.00017.

431 432 433

41. Burch, T.R.; Sadowsky, M. J.; LaPara, T.M. Air-drying beds eliminate antibiotic resistance genes and class 1 integrons in residual municipal wastewater solids. Environ. Sci. Technol. 2013, 47, 9965-9971.

434 435 436

42. Burch, T.R.; Sadowsky, M. J.; LaPara, T.M. Modeling the fate of antibiotic resistance genes and class 1 integrons during thermophilic anaerobic digestion of municipal wastewater solids. Appl. Microbiol. Biotechnol. 2016, 100, 1437-1444.

437 438 439 440

43. Beecher, N.; Crawford, K.; Goldstein, N.; Kester, G.; Lono-Batura, M.; Dziezyk, E.; Peckenham, J.; Cheng, T. Final Report: A National Biosolids Regulation, Quality, End Use, & Disposal Survey; North East Biosolids and Residuals Association: Tamworth, NH, 2007.

441 442 443

44. Burch, T.R.; Sadowsky, M. J.; LaPara, T.M. Fate of antibiotic resistance genes and class 1 integrons in soil microcosms following the application of treated residual municipal wastewater solids. Environ. Sci. Technol. 2014, 48, 5620-5627.

444 445 446

45. Baertsch, C.; Paez-Rubio, T.; Viau, E.; Peccia, J. Source tracking aerosols released from land-applied class B biosolids during high-wind events. Appl. Environ. Microbiol. 2007, 73, 4522-4531.

17

ACS Paragon Plus Environment

Page 18 of 28

Page 19 of 28

Environmental Science & Technology

447 448 449

46. Ling, A. L.; Pace, N. R.; Hernandez, M. T.; LaPara, T. M. Tetracycline resistance and class 1 integron genes associated with indoor and outdoor aerosols. Environ. Sci. Technol. 2013, 47, 4046-4052.

450 451

47. Selleck, R. E.; Collins, H. F.; Saunier, B. M. Kinetics of bacterial deactivation with chlorine. J. Environ. Eng. Div. ASCE 1978, 104, 1197-1212.

452 453

48. Crittenden, J. C.; Trussell, R. R.; Hand, D. W.; Howe, K. J., Tchobanoglous, G. Water treatment: principles and design; 2nd ed.; John Wiley & Sons: Hoboken, NJ, 2005.

454 455 456

49. Sandberg, K.; LaPara, T. M. The fate of antibiotic resistance genes and class 1 integrons following the application of swine and dairy manure to soils. FEMS Microbiol. Ecol. 2016, 92(2), 1-7; DOI: 10.1093/femsec/fiw001.

457 458

50. Aminov, R. I. The role of antibiotics and antibiotic resistance in nature. Environ. Microbiol. 2009, 11, 2970-2988.

459 460 461

51. Allen, H. K.; Donato, J.; Wang, H. H.; Cloud-Hanse, K. A.; Davies, J.; Handelsman, J. Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 2010, 8, 251-259.

462 463 464 465

52. Pruden, A.; Larsson, D. G. J.; Amezquita, A.; Collignon, P.; Brandt, K. K.; Graham, D. W.; Lazorchak, J. M.; Suzukie, Z.; Silley, P.; Snape, J. R.; Topp, E.; Zhang, T.; Zhu, Y.G. Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environ. Health Perspect. 2013, 121, 878-885.

466 467 468

53. Williams-Nguyen, J.; Sallach, J. B.; Bartelt-Hunt, S.; Boxall, A. B.; Durso, L. M.; McLain, J. E.; Singer, R. S.; Snow, D. D.; Zilles, J. L. Antibiotics and antibiotic resistance in agroecoystems: state of the science. J. Environ. Qual. 2016, 45, 394-406.

469 470

54. Sanderson, H.; Fricker, C.; Brown, R. S.; Majury, A.; Liss, S. N. Antibiotic resistance genes as an emerging environmental contaminant. Environ. Rev. 2016, 24, 205-218.

471 472 473

55. Bondarczuk, K.; Markowicz, A.; Piotrowska-Seget, Z. The urgent need for risk assessment on the antibiotic resistance spread via sewage sludge land application. Environ. Int. 2016, 87, 49-55.

474 475 476

56. Xie, W.-Y.; McGrath, S. P.; Su, J.-Q.; Hirsch, P. R.; Clark, I. M.; Shen, Q.; Zhu, Y.-G.; Zhao, F.-J. Long-term impact of filed applications of sewage sludge on soil antibioitc resistome. Environ. Sci. Technol. 2016, 50, 12602-12611.

477 478 479

57. Bae, S.; Wuertz, S. Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl. Environ. Microbiol. 2009, 75, 29402944.

480 481

58. Marti, R.; Tien, Y.-C.; Murray, R.; Scott, A.; Sabourin, L.; Topp, E. Safely coupling livestock and crop production systems: How rapidly do antibiotic resistance genes

18

ACS Paragon Plus Environment

Environmental Science & Technology

482 483

dissipate in soil following a commercial application of swine or dairy manure? Appl. Environ. Microbiol. 2014, 80, 3258-3265.

484

59. Mazel, D. Integrons: agents of bacterial evolution. Nat. Rev. Microbiol. 2006, 4, 608-620.

485 486

60. Gillings, M. R.; Paulsen, I. T.; Tetu, S. G. Ecology and evolution of the human microbiota: fire, farming, and antibiotics. Genes 2015, 6, 841-857.

487 488 489

61. Guo, X.; Xia, R.; Han, N.; Xu, H. Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China. Lett. Appl. Microbiol. 2011, 52, 667-675.

490 491 492

62. Stalder, T.; Barraud, O.; Casellas, M.; Dagot, C.; Plot, M.-C. Integron involvement in environmental spread of antibiotic resistance. Front. Microbiol. 2012, 3(119), 1-14; DOI: 10.3389/fmicb.2012.00119

493 494 495 496

63. Gatica, J.; Tripathi, V.; Green, S.; Manaia, C.; Berendonk, T.; Cacace, D.; Merlin, C.; Kreuzinger, N.; Schwartz, T.; Fatta-Kassinos, D.; Rizzo, L.; Schwermer, C. U.; Garelick, H.; Jurkevitch, E.; Cytryn, E. High throughput analysis of integron gene cassettes in wastewater environments. Environ. Sci. Technol. 2016, 50, 11825-11836.

497 498 499

64. Gillings, M. R.; Gaze, W. H.; Pruden, A.; Smalla, K.; Tiedje, J. M.; Zhu, Y.-G. Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution. ISME J 2015, 9, 1269-1279.

500 501 502

65. Ma, L.; Li, A.-D.; Yin, X.-L.; Zhang, T. The prevalence of integrons as the carrier of antibiotic resistance genes in natural and man-made environments. Environ. Sci. Technol. 2017, 51, 5721-5728.

503 504 505

66. Zhang, Y.; Snow, D.; Parker, D.; Zhou, Z.; Li, X. Intracellular and extracellular antimicrobial resistance genes in livestock waste management structures. Environ. Sci. Technol. 2013, 47, 10206-10213.

506 507 508

67. Mao, D. Q.; Luo, Y.; Mathieu, J.; Wang, Q.; Feng, L.; Mu, Q. H.; Feng, C. Y.; Alvarez, P. J. J. Persistenc of extracellular DNA in river sediment facilitates antibiotic resistance propagation. Environ. Sci. Technol. 2014, 48, 71-78.

509 510 511

68. Ghosh, S.; LaPara, T. M. The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J 2007, 1, 191-203.

512 513 514

69. Nesme, J.; Cecillon, S.; Delmont, T. O.; Monier, J. M.; Vogel, T. M.; Simonet, P. Largescale metagenomic-based study of antibiotic resistance in the environment. Curr. Biol. 2014, 24, 1096-1100.

515 516 517

70. Bengtsson-Palme, J.; Boulund, F.; Fick, J.; Kristiansson, E.; Larsson, D. G. J. Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Front. Microbiol. 2014, 5, 1-14

19

ACS Paragon Plus Environment

Page 20 of 28

Page 21 of 28

Environmental Science & Technology

518

20

ACS Paragon Plus Environment

Environmental Science & Technology

Page 22 of 28

Table 1. Quantities of genes encoding 16S rRNA, five different antibiotic resistance determinants, and the integrase of class 1 integrons in wastewater solids treated in different ways prior to being applied to soil microcosms. Quantities are presented as the arithmetic means (± standard deviation; n = 3) of log10-transformed gene copies per gram of wet sludge. No Treatment

Air dried

Aerobic Anaerobic Anaerobic Anaerobic Anaerobic Alkaline Pasteurization Digestion Digestion Digestion Digestion Digestion Stabilization (17°°C) (38°°C) (55°°C) (63°°C) (69°°C)

16S rRNA

9.4 ± 0.2

9.2 ± 0.4

9.8 ± 0.1

9.4 ± 0.3

9.5 ± 0.1

9.3 ± 0.1

9.0 ± 0.1

10.2 ± 0.03

10.1 ± 0.05

erm(B)

7.5 ± 0.5

4.9 ± 1.1

6.7 ± 0.1

6.7 ± 0.3

6.0 ± 0.2

6.3 ± 0.2

6.6 ± 0.1

8.8 ± 0.05

8.9 ± 0.05

intI1

7.2 ± 0.2

6.8 ± 0.4

8.3 ± 0.1

6.7 ± 0.2

6.6 ± 0.1

6.8 ± 0.1

6.4 ± 0.1

8.8 ± 0.03

8.9 ± 0.03

sul1

7.6 ± 0.1

6.6 ± 0.4

8.0 ± 0.1

7.0 ± 0.3

6.5 ± 0.1

6.6 ± 0.1

6.2 ± 0.1

9.2 ± 0.07

8.7 ± 0.02

tet(A)

6.0 ± 0.2

4.3 ± 1.0

7.2 ± 0.1

5.5 ± 0.3

5.8 ± 0.1

5.8 ± 0.1

5.4 ± 0.1

7.4 ± 0.01

7.7 ± 0.04

tet(W)

7.3 ± 0.2

4.6 ± 0.3

6.2 ± 0.1

6.5 ± 0.3

6.4 ± 0.1

6.4 ± 0.2

6.5 ± 0.1

8.1 ± 0.07

8.4 ± 0.03

tet(X)

5.5 ± 0.3

5.6 ± 0.5

8.0 ± 0.1

4.3 ± 0.4

4.1 ± 0.1

4.3 ± 0.1

3.9 ± 0.3

7.4 ± 0.1

7.6 ± 0.02

21

ACS Paragon Plus Environment

Page 23 of 28

Environmental Science & Technology

Table 2. Kinetic coefficients regressed when the decay of ARGs and intI1 were fit to a modified CollinsSelleck model (ΛCS, b) and a first-order model (k). n.d. = not determined due to a poor fit to the CollinsSelleck model or due to insufficient data associated with gene levels below the quantification limit.

Gene

Treatment

ΛCS

b (d)

k (d)

t1/2

erm(B)

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

-1.6 n.d. -1.3 -1.6 n.d. n.d. n.d. -1.4 -1.0

0.3 n.d. 16 0.3 n.d. n.d. n.d. 0.9 0.07

0.02 n.d. 0.01 0.02 n.d. n.d. n.d. 0.02 0.01

41 n.d. 49 30.2 n.d. n.d. n.d. 46 64.8

intI1

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

-1.0 -0.5 -0.8 -0.8 -0.3 -0.2 -0.6 -0.4 -0.4

15 21 6.4 93 1.8 0.2 14 0.003 0.2

0.01 0.006 0.008 0.009 0.004 0.002 0.007 0.005 0.004

52 124 86 75 187 330 99 151 169

sul1

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization

-0.8 -0.5 -0.7 -0.9 -0.3 -0.07 -0.6 -0.7

10 20 10 27 0.03 500 103 90

Alkaline stabilization

-0.5

0.02

0.005

151

tet(A)

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

-1.1 n.d. -0.5 -1.3 n.d. n.d. n.d. -0.2

0.4 n.d. 5.3 230 n.d. n.d. n.d. 1.4

0.01 n.d. 0.006 0.02 n.d. n.d. n.d. 0.002 0.003

60 n.d. 122 46 n.d. n.d. n.d. 408 203

tet(W)

No Treatment

-1.3

16.6

0.01

60

22

ACS Paragon Plus Environment

Environmental Science & Technology

tet(X)

Page 24 of 28

Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

n.d. -1.4 -0.2 n.d. n.d. n.d.

n.d. 8.0 1.4 n.d. n.d. n.d.

n.d. 0.015 0.002 n.d. n.d. n.d.

n.d. 47 346 n.d. n.d. n.d.

-0.2 -0.3

< 0.001 < 0.001

0.004 0.003

182 203

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

-2.1 -0.7 -1.6 n.d. n.d. n.d. n.d. n.d. n.d.

6.1 65.5 17.8 n.d. n.d. n.d. n.d. n.d. n.d.

0.02 0.008 0.02 n.d. n.d. n.d. n.d. n.d. n.d.

30 87 39 n.d. n.d. n.d. n.d. n.d. n.d.

23

ACS Paragon Plus Environment

Page 25 of 28

Environmental Science & Technology

Table 3. Quantities of genes encoding 16S rRNA, five different antibiotic resistance determinants, and the integrase of class 1 integrons in soils to which wastewater solids had been applied and then subsequently incubated for 6 months. Wastewater solids were treated in different ways prior to being applied to soil microcosms. Quantities are presented as the arithmetic means (± standard deviation; n = 3) of log10-transformed gene copies per gram of wet soil. Superscripted letters denote groups that statistically clustered with each other (P < 0.05). All gene quantities were below the detection limit quantities in the control soil microcosms (to which no wastewater solids were applied) for all genes except for 16S rRNA genes (7.7 ± 0.1). b.d. = below detection.

No Treatment

Air drying

Aerobic Digestion (17°°C)

Anaerobic Digestion (38°°C)

Anaerobic Digestion (55°°C)

Anaerobic Digestion (63°°C)

Anaerobic Digestion (69°°C)

Pasteurization

Alkaline Stabilization

16S rRNA

8.0 ± 0.2A

7.4 ± 0.03B,C

7.7 ± 0.04A,B

7.7 ± 0.5A,B

7.5 ± 0.1B,C

7.7 ± 0.02A,B

7.8 ± 0.1A,B

7.1 ± 0.2C

7.1 ± 0.03C

erm(B)

3.2 ± 0.2B

b.d.

4.0 ± 0.1A

b.d.

b.d.

b.d.

b.d.

3.0 ± 0.1B

3.1 ± 0.1B

intI1

3.4 ± 0.01C,D

5.1 ± 0.4A

5.0 ± 0.1A

4.3 ± 0.1B

3.7 ± 0.1C,D

3.9 ± 0.1B,C

3.3 ± 0.1D

3.9 ± 0.4B,C,D

3.9 ± 0.01B,C

sul1

4.6 ± 0.1B,C

5.7 ± 0.5A

5.0 ± 0.04A,B

4.1 ± 0.01B,C,D

3.2 ± 0.1D,E

3.6 ± 0.1D,E

2.9 ± 0.4E

3.7 ± 0.8C,D,E

3.5 ± 0.1D,E

tet(A)

b.d.

b.d.

4.2 ± 0.1A

4.0 ± 0.3A

b.d.

b.d.

b.d.

b.d.

2.8 ± 0.1B

tet(W)

4.3 ± 0.01A

b.d.

2.8 ± 0.1B

b.d.

b.d.

2.4 ± 0.1C

2.2 ± 0.1C

b.d.

b.d.

tet(X)

4.5 ± 0.03A

4.1 ± 0.6A

4.6 ± 0.1A

b.d.

b.d.

b.d.

b.d.

b.d.

b.d.

24

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 1. Quantities of ARGs and intI1 following the application of untreated wastewater solids to soil. Solid circles represents individual qPCR quantifications performed in triplicate from each of triplicate microcosms. The solid line represents the best fit to a modified Collins-Selleck model; the dashed line represents the best fit to a first-order kinetic model, excluding the initial time point. The regressed coefficients for the kinetic models are shown in Table 2.

25

ACS Paragon Plus Environment

Page 26 of 28

Page 27 of 28

Environmental Science & Technology

Figure 2

Figure 2. The quantities of sul1 genes following the application of treated wastewater solids to soils. Solid circles represents individual qPCR quantifications performed in triplicate from each of triplicate microcosms. The solid line represents the best fit to a modified Collins-Selleck model; the dashed line represents the best fit to a first-order kinetic model, excluding the initial time point. The regressed coefficients for the kinetic models are shown in Table 2. Analogous plots for the quantities of erm(B) (Fig. S1), intI1 (Fig. S2), tet(A) (Fig. S3), tet(W) (Fig. S4), and tet(X) (Fig. S5) are provided in the Supporting Information.

26

ACS Paragon Plus Environment

Environmental Science & Technology

TOC graphic 338x190mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 28 of 28