Influence of Manure Application on the Environmental Resistome

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Influence of Manure Application on the Environmental Resistome Under Finnish Agricultural Practice with Restricted Antibiotic Use Johanna Muurinen, Robert D. Stedtfeld, Antti Karkman, Katariina Pärnänen, James M. Tiedje, and Marko P.J. Virta Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 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 35

Environmental Science & Technology

4

Influence of Manure Application on the Environmental Resistome Under Finnish Agricultural Practice with Restricted Antibiotic Use

5

Johanna Muurinen†*, Robert Stedtfeld‡, Antti Karkman§, Katariina Pärnänen†, James Tiedje‡ and

6

Marko Virta†*

7 8

† University of Helsinki, Department of Food and Environmental Sciences, Division of

9

Microbiology and Biotechnology, Viikinkaari 9, 00014 University of Helsinki, Finland

10

‡ Center for Microbial Ecology, Department of Civil and Environmental Engineering, Michigan

11

State University, East Lansing, MI 48824-1325, USA

12

§ University of Helsinki, Department of Biosciences, Viikinkaari 1, 00014 University of

13

Helsinki, Finland.

14

*Corresponding authors contact information:

15 16 17 18 19 20 21 22 23

Marko Virta: Phone: +358 294157586 Mobile: +358 504480000 E-mail: [email protected]

1 2 3

Johanna Muurinen: Phone: +358 4652249 E-mails: [email protected], [email protected]

24 25

ACS Paragon Plus Environment


26

Abstract

27

The co-occurrence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in

28

farm environments can potentially foster the development of antibiotic resistant pathogens. We

29

studied the resistome of Finnish dairy and swine farms where use of antibiotics is limited to

30

treating bacterial infections and manure is only applied April to September. The resistome of

31

manure, soil and tile drainage water from ditch was investigated from the beginning of the

32

growing season until forage harvest. The relative ARG and MGE abundance was measured using

33

a qPCR array with 363 primer pairs. Manure samples had the highest abundance of ARGs and

34

MGEs, which increased during storage. Immediately following land application, the ARGs

35

abundant in manure were detected in soil, but their abundance decreased over time with many

36

becoming undetectable. This suggests that increases in ARG abundances after fertilizing are

37

temporary and occur annually under agricultural practices that restrict antibiotic use. A few of

38

the ARGs were detected in the ditch water but most of them were undetected in the manure. Our

39

results document the dissipation and dissemination off-farm of ARGs under Finnish limited

40

antibiotic use, and suggest that such practices could help reduce load of antibiotic resistance

41

genes in the environment.

42 43


Page 2 of 35

Page 3 of 35


44

Introduction

45

Agricultural antibiotic use is suspected to be an important contributor in the increasing

46

emergence of infections caused by antibiotic resistant bacteria1-3. Intriguingly, many antibiotic

47

resistance genes (ARGs) are carried by environmental microorganisms and existed before the

48

antibiotic era4, 5. Production animal farms are proposed to act as reservoirs where genetic

49

material from environmental bacteria might transfer to human or animal associated bacteria6,

50

7

including zoonotic pathogens1, 2, 8-11.

51

ARGs are often associated with integrons and mobile genetic elements (MGEs), such

52

as insertion sequences, transposons and plasmids, which can capture genetic material from the

53

environment and transfer between bacterial species7, 12-14. Horizontal gene transfer occurs

54

frequently in the animal gut15, but also agricultural soils can act as hotspots for bacterial

55

exchange of genetic material7, 16.

56

Use of antibiotics in agriculture can select for antibiotic resistant bacteria in the animal

57

intestinal microbiome17-20, in the manure storage areas and when the manure is applied to land as

58

fertilizer16,17. Furthermore, in soil the intestinal bacteria and their resistance determinants mix

59

with soil bacteria, which also harbor ARGs4, 5, 21, providing additional genetic material for

60

evolutionary adaptation. During harvest, bacteria from manure and soil may end up in harvested

61

fodder crops together with dust and small soil particles. Thus, ARGs present in the soil during

62

harvest may be cycled back into the animal gut. Moreover, intrinsic soil bacteria carrying ARGs

63

and MGEs might contribute to the animal gut resistome via fodder ingestion.

64

Production animal farms have been studied widely for existence of ARGs and traces of

65

antibiotic residues16, 20, 22, 23, aiming to determine whether the use of antibiotics and land

66

application of manure changes the farm environment’s natural resistome; ARG abundances have

67

been shown to change and increase due to farming24. Critical examination of the farming



68

practices in animal production might reveal procedures, which could be modified to decrease the

69

enrichment and dissemination of antibiotic resistance.

70

We recently showed25 that despite low antibiotic use, ARGs disseminate to soils as a

71

consequence of manure application and that ARG abundance increases in manure during winter

72

storage, a stage necessary for Finnish climate conditions. However, since we quantified only

73

three ARGs associated to human impacted environments, we were uncertain if our observations

74

hold true for other ARGs as well. Furthermore, ranking the risks from environmental ARGs

75

requires the evaluation of mobility potential26. Due to possible transfer of bacteria and ARGs

76

from the manured fields to the animal gut via the fodder, it is important to follow the soil

77

resistome through fodder harvest. It is equally important to clarify whether farming practices

78

disseminate ARG pollution further by leaching or surface water run-off from the fields.

79

In the present study we followed the same swine and dairy farms, but instead of

80

assaying three ARGs we quantified many more genes related to antibiotic resistance and transfer

81

with 363 primer pairs using a high throughput qPCR array22, 27. Samples were taken from fresh

82

manure, stored manure, unfertilized soil, soil fertilized with manure, and tile drainage water from

83

ditch, which was sampled before and after land application. We aimed to answer the following

84

questions: do ARGs disseminate to the environment (field soil and surface waters), are ARGs

85

enriched in stored manure and are ARG abundances elevated in soils at crop harvesting time. For

86

evaluating the ARG mobility potential, genes related to MGEs were also quantified. Our results

87

indicated dissemination of resistance determinants to soils immediately following manure

88

application, but the abundance and richness of the disseminated genes decreased substantially by

89

6 weeks after land application. We also showed that storing the manure increased relative

90

abundance of several ARGs and MGEs.


Page 4 of 35

Page 5 of 35


91

Materials and Methods

92

Study sites, sampling and DNA extraction

93

The study sites were two dairy farms (T1 and T2) and two swine farms (T3 and T4).

94

Finnish agricultural practices used at these farms were detailed previously25. Farms, farming

95

practices, soil textures, animal numbers, and the antibiotics used are presented in Table 1. All the

96

sampled soils had been in regular agricultural use for several years and fertilized with manure

97

annually. Samples collected from each farm included: fresh manure from inside the animal

98

shelter (I), stored manure from the open-air lagoon or silo (M), unfertilized soil before the

99

application of the manure (SB), fertilized soil taken on the same day as manure was applied

100

(SA), soil 2 and 6 weeks after fertilization from the same field sites (S2WA and S6WA,

101

respectively), tile drainage water from ditch (ditch water) taken while manure was applied to the

102

field (D) and after 2 and 6 weeks (D2WA and D6WA, respectively). Sampling of the soil and

103

manure was described previously25. Water samples were collected from the ditch water below

104

the end of the drainage tile coming from the studied field. Samples were collected in 1 L factory

105

clean plastic bottles. Triplicate biological replicates were collected from all the farms and sample

106

sites.

107

Samples were transported to the laboratory on ice in a Styrofoam cooler. Soil and

108

manure samples were sieved by hand through a 5-mm screen. After homogenization soil and

109

manure samples were stored in −20°C before DNA extraction. Ditch water samples (350 mL)

110

were filtered through a 0.2 µm pore size mixed cellulose ester membrane filter (GE Healthcare

111

Life Sciences) and the filters were stored in −20°C before DNA extraction. DNA was extracted

112

from the manure, soil and water samples with PowerLyzer PowerSoil and PowerWater DNA

113

Isolation Kits (MO BIO Laboratories) according to the manufacturer’s protocols. Extracted DNA

114

was stored in −20°C before qPCR analysis.



115

Quantitative PCR and primers

116

Quantitative PCR reactions were conducted using WaferGen SmartChip Real-time

117

PCR system as reported previously28, with the exception that the threshold cycle (Ct) of 27 was

118

used as a cut-off between true positive and primer-dimer amplification22, 27. Briefly, 5,184

119

parallel 100 nL reactions were dispensed with the SmartChip Multisample Nanodispenser

120

(WaferGen Biosystem, Freemont, CA, USA) to the SmartChip (WaferGen Biosystem, Freemont,

121

CA, USA). PCR cycling conditions and raw data processing were conducted as previously

122

described by Wang et al28. The qPCR reactions were performed using 370 primer sets (assays)

123

(Table S1) as described in previous studies27-30. Reference sequences for primers were obtained

124

using Functional Gene Pipeline and repository31 with the same design parameters and protocol as

125

previously described32. Seven of the 370 primer sets targeted different housekeeping genes but

126

the16S rRNA gene (universal primers) was the only housekeeping gene detected in all samples

127

and therefore was used for normalization. The rest of the primer sets targeted genes related to

128

antibiotic resistance, which were either ARGs, genes conferring resistance to other antibacterial

129

agents or genes related to mobile genetic elements (MGEs).

130

Data analysis

131

All data analysis was done with R version 3.2.333 and RStudio Version 0.98.110334.

132

The ∆Ct values, ∆∆Ct values, relative gene abundances (R) and fold changes (FC) were

133

calculated as follows:

134

∆Ct = Ct(ARG)- Ct(16S)

135

∆∆Ct = ∆CtTreatment sample − ∆CtReference sample

136

R = 2-∆Ct

137

FC = 2−∆∆CT

138

where Ct is the threshold cycle, ARG denotes one of the 363 antibiotic resistance gene

139

assays and 16S the 16S rRNA gene assay, Treatment sample is the experimental sample (e.g.


Page 6 of 35

Page 7 of 35


140

fertilized soil) and Reference sample is the sample taken before the treatment (e.g. unfertilized

141

soil). Genes under the detection limit were given a ∆Ct value of 20, which was higher than any

142

observed ∆Ct (15.6). Relative gene abundances (R) were log10 transformed for statistical

143

analysis.

144

Figures 1–3, S3-S5 and S1 were produced with packages ggplot235 and gplots36,

145

respectively. Non-linear multidimensional scaling and environmental fitting were done using

146

Bray-Curtis dissimilarity index with functions metaMDS and envfit from package vegan37. For

147

analyzing co-occurrences, a correlation matrix between ARG and MGE abundances in all

148

manure, soil and water samples within dairy farms (T1 & T2) and swine farms (T3 & T4) was

149

produced. Correlations between ARGs and MGEs and resultant p-values for network analysis

150

were obtained with package psyhc38 using False discovery rate control39. Only ARG and MGE

151

pairs with significant Spearman’s correlation of ρ > 0.8 were approved. In addition, the

152

relationship between these pairs was inspected visually and pairs with correlation created by

153

outliers were discarded. The network analysis was built with Gephi version 0.9.140 having equal

154

weights and sizes for all the edges and nodes, respectively.

155

The effect of environmental variables on the relative abundance of genes was modeled

156

with permutational multivariate analysis of variance by function adonis from vegan package41.

157

The compared samples were fresh manure to stored manure, unfertilized soil against soil 2 and 6

158

weeks after fertilization and ditch water against samples taken 2 and 6 weeks later (Table 2).

159

Student’s t-test for paired samples was used to test the significance of fold changes (FC) using

160

False discovery rate control39.

161 162 163



164

Results

165

Diversity of ARGs and MGEs

166

In total 182 out of 363 ARG and MGE qPCR assays were positive in one or more

167

samples. Out of the positive assays, 161 targeted ARGs and 21 MGEs. Fresh manure had the

168

highest diversity of ARGs and MGEs with 130 positive assays, followed by stored manure and

169

manured soils (Figure 1). The number of positive assays decreased in fertilized soil between the

170

2- and 6-week sampling points (Figure 1). Only 29 assays were positive in unfertilized soil

171

samples. Water samples collected from ditches receiving tile drainage water from the fields had

172

the lowest diversity of ARGs and MGEs (Figure 1). Twenty-one genes detected in fresh manure

173

were absent in stored manure, and 19 genes found in stored manure were not detected in fresh

174

manure (Figure S1A). Unfertilized soil samples shared 13 genes with stored manure used in

175

fertilization (Figure S1B). The genes detected in all samples covered all three major resistance

176

mechanisms, with tetracycline, Macrolide− Lincosamide− Streptogramin B (MLSB) and

177

multidrug efflux being the three most common resistance gene groups (Figure S2).

178 179 180

Abundances and dissemination of ARGs and MGEs ARGs and MGEs were quantified relative to the 16S rRNA gene abundance in the

181

samples. Manure had the highest relative abundances of ARGs and these manure-associated

182

ARGs were not detected in unfertilized soil or ditch water sampled before fertilization (Figure

183

2). Likewise ARGs abundant in unfertilized soil or in ditch water were not abundant or even

184

detected in manure (Figure 2). After fertilization, the manure associated ARGs and MGEs were

185

present in soil, hence a consequence of fertilization. However, the abundance and number of

186

these ARGs and MGEs clearly decreased from fertilized soil to fertilized soil samples taken 2

187

and 6 weeks after manure application (Figure 2, Figure S1B). Six manure associated ARGs and

188

MGEs were found in ditch water sampled after fertilization, indicating possible dissemination to


Page 8 of 35

Page 9 of 35


189

leachate water (Figure 2, Figure S1C). Most of these genes were MGEs discovered on farms T1

190

and T4 (Figure 2).

191

ARGs belonging to most resistance groups were detected from all of the sampled

192

environments, although resistance genes against sulfonamides, tetracyclines (except tet-G),

193

trimethoprim and disinfectants were not found in ditch water (Figure 2, Figure S3). Genes

194

encoding resistance against aminoglycosides, disinfectants, MLSB, tetracyclines, sulphonamide,

195

trimethoprim or vancomycin were common in manure and fertilized soils (Figure 2, Figure S3).

196

On the contrary, genes conferring resistance to β-lactams and genes classified as multidrug

197

resistance were mainly detected in unfertilized soil, in ditch water, and rarely found in manure

198

(Figure 2, Figure S3). Genes from all MGE groups were present in all samples, while plasmids

199

were more abundant in ditch water and in unfertilized soil, than in manure (Figure 2, Figure S3).

200

Integrons, insertion sequences and transposases were more prevalent in manure than in

201

unfertilized soil or ditch water (Figure 2, Figure S3).

202

Changes in ARG and MGE abundances due to storage and fertilization

203

During manure storage the relative abundance increased more than four-fold for 41

204

genes, and more than two-fold for 62 genes (Figure 3, Figure S4, Table S2). The highest increase

205

(up to 65-fold) was observed in tetracycline resistance genes, followed by sulphonamide and

206

aminoglycoside resistance genes with up to 45-fold and 41-fold increases (Figure 3, Figure S4,

207

Table S2).

208

Manure fertilization increased the relative abundance of ARGs and MGEs in soil. The

209

increase was more than two-fold in 62 observations in soil 2 weeks after the fertilization

210

compared to unfertilized soil (Table S2, Figure S4). The highest significant increases in soil 2

211

weeks after land application included aminoglycoside resistance genes and integrons (Figure S4,

212

Table S2). In soil samples taken 6 weeks after fertilization, the highest significant enrichment



213

compared to unfertilized soil was in the relative abundance of blaOXY (80-fold) (Figure 3, Table

214

S2).

215

Six weeks after fertilization, some of the manure-associated MGEs and genes

216

conferring resistance against aminoglycosides, disinfectants and vancomycin were still elevated

217

in soil. Nevertheless, most of the genes enriched in soil 6 weeks after fertilization were already

218

present in unfertilized soil (Figure 2, Figure 3, Figure S5). The relative abundance of manure-

219

associated genes in soil 6 weeks after fertilization decreased compared to the soil 2 weeks after

220

fertilization. A more than fourfold decrease in the relative abundances of ARGs and MGEs was

221

observed in 34 genes and more than two-fold decrease in 46 genes (Figure 3, Figure S5) over the

222

4 weeks. The decrease was also statistically significant for 30 genes, varying from 200- to 9000-

223

fold (Table S2).

224

The relative ARG and MGE abundances also increased in ditch water sampled 2- and 6

225

weeks after the fertilization, compared to their initial concentrations (Figure S5). However,

226

among the enriched genes, only intI2-02 and tnpA/IS21 were detected also in manure samples

227

and had statistically significant fold increases (Figure S5, Table S2).

228

Factors affecting ARG and MGE profiles

229

In ordination analysis soil samples taken immediately after fertilization were most

230

similar to the centroid of manure samples, whereas soil samples taken before fertilization and

231

soil samples taken 6 weeks after were closer to the centroid of ditch water samples (Figure 4).

232

The effect of environmental variables on the relative abundance of genes was studied with

233

permutational multivariate analysis of variance (Table 2). Sample type explained most of the

234

variance (R2=0.39; p 0.8) significant (p < 0.05)

247

correlations in the two farm types was studied with network analysis across all the manure, soil

248

and water samples. All the detected ARGs and MGEs are shown in Figure S3. The network

249

analysis showed that the ARGs detected in manure and fertilized soils co-occurred with

250

integrons, insertion sequences and transposases (Figure 5). In addition, most of the ARGs that

251

enriched in stored manure co-occurred with MGEs (Figure 3, Figure 5, Figure S4). The ARGs

252

and MGEs that were abundant in soil before fertilization and in ditch water (Figure 2, Figure

253

S3), co-occurred mainly with plasmids (Figure 5). The insertion sequences IS1133, IS613 and

254

ISAba3 had somewhat different network in dairy farms verses swine farms, indicating that there

255

are some differences between ARG and MGE dynamics in animal gut and manure dependent on

256

animal species (Figure 5). The network of dairy farms had two genes encoding clinically

257

relevant β-lactamases co-occurring with MGEs, however, less genes conferring resistance

258

against MLSBs co-occurring with MGEs than in the network of swine farms (Figure 5). The

259

vancomycin resistance genes were mainly detected in swine manure (Figure 2, Figure S3) and

260

are associated with mobile elements (Figure 5).

261 262



263

Discussion

264

Animal manure and farm environments are considered ARG reservoirs, which could

265

transfer resistance to human and animal pathogens1, 3, 6. Our results are consistent with this risk,

266

as 182 of the analyzed 363 assays targeting ARGs and MGEs were positive. However, samples

267

taken from soil before the annual fertilization in spring did not show high diversity of ARGs or

268

MGEs, although these soils have fertilized annually with manure for decades. Insertion

269

sequences and class 1 integrons, which are considered to be markers of ARG-contamination in

270

environmental samples41, were not detected in unfertilized soil. Moreover, the ARG profiles of

271

unfertilized soil samples were similar to non-fecal contaminated soils29. This might be due to the

272

annual freeze-thaw cycle of soils and Finnish agricultural practices, which limit manure

273

application to only the April-May to September-October time period and enforce prudent

274

antibiotic use. In the European Union, use of antimicrobials for growth promotion is not allowed

275

in food animals and in Finland antibiotics are predominantly used to medicate animals suffering

276

from bacterial infections. In addition, mainly narrow-spectrum antibiotics are used on Finnish

277

farms42. These practices could lead to a lower initial proportion of resistant bacteria in manure

278

compared to countries where antibiotics are used non-therapeutically or prophylactically.

279

Indeed, the extensive use of antibiotics has been shown to increase the diversity and abundance

280

of ARGs in soil on Chinese swine farms22. While we don’t know if the restricted antibiotic use

281

in Finland has actually lowered the environmental ARG load since we had no high antibiotic use

282

control, our study does, however, document the antibiotic resistant gene types, abundances,

283

dissipation and dissemination off-farm under this restricted use policy.

284

As expected, genes abundant in manure were transferred to soil as a consequence of

285

manure fertilization. These manure-associated genes confer resistance mainly to

286

aminoglycosides, disinfectants, MLSBs, tetracyclines and sulfonamides and may be linked to the

287

co-occurring mobile genetic elements. Importantly, the abundance and diversity of manure-


Page 12 of 35

Page 13 of 35


288

associated ARGs and MGEs clearly decreased 6 weeks after manure application. This suggests

289

that bacteria originating from manure carrying most of these genes are not well adapted to the

290

soil environment. However, some of the ARGs and MGEs were still detectable in soil 6 weeks

291

after manure application, but their relative abundances decreased significantly since manure

292

application. Since these persisting ARGs and MGEs were not detected in the unfertilized soil

293

samples (which had been manured on previous growing season), and their abundance was

294

decreasing with time, it is likely that they will be below detection limit by winter. Thus the

295

results indicate that the manure application does not permanently change the resistance profile of

296

these soils, but the changes in the resistome are cyclic depending on season, time and amount of

297

manure application. In contrast, Schmitt et al43 found that Swiss farmland, previously exposed to

298

intensive farming and fertilized with manure of animals that were routinely treated with

299

chlortetracycline, sulfamethazine, and tylosin, harbored multiple resistance determinants and

300

resistant organisms even years after the last manure application. In addition to exposure to

301

antibiotics, fecal contamination has been linked to higher prevalence of resistance genes in

302

various environments44,45,30. Since the soils before fertilization in our study harbored very low

303

initial proportions of resistance genes related to intensive farming, we suggest that adopting

304

similar agricultural practices to those in Finland might help decrease the prevalence of clinically

305

relevant ARGs and MGEs in agricultural soils.

306

The most prevalent genes in unfertilized soil and in ditch water encode β-lactamases or

307

multidrug resistance efflux-pumps. These ARGs were not abundant in manure and likely belong

308

to the intrinsic environmental resistome. β-lactamase genes and genes encoding multidrug

309

resistance are widely dispersed in environments with limited human impact5, 46, 47. While others

310

have shown genes encoding β-lactamases can be increased in soil as a consequence of manure

311

treatment 48, 49, our results did not show an increase in the relative abundance of these genes in

312

fertilized soils. Recently, a metallo-β-lactamase gene blaIMP-27, on an IncQ1 plasmid carried by



313

several bacteria, was detected first time in a swine farm in US50. We detected blaIMP genes from

314

unfertilized soil and ditch water. The cycle of bacteria between the farm environment and farm

315

animals, along with use of selective antibiotics and presence of MGEs, increases the probability

316

for transfer of resistance genes from environmental bacteria to bacteria capable of causing

317

infections.

318

Unfertilized soil harbored plasmids but not other MGE types. Only a few plasmid

319

targeting assays were positive in our results. This indicates that these plasmids and their

320

preferred hosts are not prevalent in manure samples and only a few of them are prevalent in the

321

farm environment. Another possibility is that the primers targeting plasmids in our assays were

322

not suitable for plasmid sequences in most of our samples. According to the network analysis,

323

some of the ARGs detected from unfertilized soil or ditch water co-occur with plasmids. As

324

pointed out by Götz et al.51, the presence of plasmids in the farm environment indicates that

325

these surroundings have capacity for mobilizing ARGs.

326

Our previous results indicated that the relative abundance of ARGs increased during

327

manure storage25. We confirmed the increase in the present study with multiple genes.

328

According to statistical analysis, the number of animals on farms and animal species explained

329

differences in ARG abundance in manure samples, however, these variables are probably not

330

causing the increase in the relative abundance observed in the stored manure. Our network

331

analysis suggested that many of the enriched ARGs are incorporated with MGEs. The MGEs co-

332

occurring with the enriched ARGs have been shown to carry these ARGs also in various other

333

studies: insertion sequences encoding transposases have been linked to tetracycline resistance

334

genes52–54 and conjugative transposons to aminoglycoside, macrolide and tetracycline resistance

335

genes55-59. qacE∆1, sul1 and intI, which are part of the conserved regions of class I integrons13, 60,

336

61

337

class 1 integrons60, 62, 63.

, were co-enriched along with aadA, which are also commonly found in the gene cassettes of


Page 14 of 35

Page 15 of 35

338


The observed increase in the relative abundance of certain ARGs in the stored manure

339

might arise due to horizontal gene transfer, since MGEs found in manure co-occurred with

340

ARGs. Another possibility is that the enrichment of ARGs and MGEs occurs as a result of a shift

341

in the bacterial community from the fresh manure to a bacterial community that is better adapted

342

to the conditions in the storage silo or lagoon64 Nevertheless, the drivers causing the increase of

343

relative abundance of ARGs in stored manure should be clarified, since the phenomenon has

344

been noticed also in treatment of wastewater biosolids in US65. Using e.g. epicPCR66 to

345

determine which species carry these ARGs and MGEs would help in understanding the reason

346

for increased abundance of ARGs during manure storage.

347

Antibiotic residues could also explain the enrichment of certain genes in the stored

348

manure. However, we did not detect traces of antibiotics from stored manure in our previous

349

study25. Since the samples were collected from the same farms for this study, it is likely that the

350

concentrations of the trace antibiotics would have been below the detection limit. Nevertheless,

351

during the time manure was collected some animals on the farms were medicated with

352

antibiotics and therefore traces of antibiotics might have an effect on the ARG abundance in the

353

stored manure. It is known that as a response to sub-inhibitory concentrations of antibiotics,

354

bacteria can increase their genetic material exchange rates and promote horizontal dissemination

355

of resistance genes66-71. Moreover, the sub-inhibitory concentrations may also cause selection

356

pressure72. In addition to antibiotic traces, environmental conditions can induce horizontal gene

357

transfer due to stress response mechanisms68, 73. Cold stress has been shown to increase relative

358

abundances of ARGs in wastewater, possibly due to induced horizontal gene transfer65. During

359

winter the temperature of manure in the storage silo or lagoon can be below 5°C in countries

360

with boreal climate. It is possible that cold stress and antibiotic residue induced horizontal gene

361

transfer could contribute to the enrichment of ARGs in addition to the community shifts. It must



362

also be noted that we cannot differentiate viability of the ARGs detected by the DNA-based

363

qPCR array used in this study.

364

All the sampled environments harbored their characteristic ARGs. Forsberg et al.73

365

observed that nitrogen fertilization favored particular organisms resulting in enrichment of

366

certain ARG families (ABC-transporters) and depletion of others (β-lactams). Alterations in

367

community composition may have caused some of the changes in the ARG profiles in this study.

368

Few of the ARGs prevalent in unfertilized soil samples were less prevalent in samples taken

369

soon after manure application. However, their abundance increased again at the forage

370

harvesting time. It is likely that the abundance of intrinsic soil ARGs decreased as a result of

371

additional bacterial load from the manure and the change of soil conditions.

372

The soil microbial community in the studied farms was resilient to manure loads, since

373

the abundance and diversity of manure-associated resistance determinants clearly decreased only

374

6 weeks after the manure application and these genes were not detected in unfertilized soil. The

375

large majority of genes abundant in manure did not spread to the receiving waters, which means

376

that the studied farms are probably not widely disseminating a large load of ARGs and MGEs.

377

However, the threat arising from the combination of natural resistome and MGEs and the

378

possible enrichment of resistance determinants during storage of manure used as land applied

379

fertilizer should not be underestimated.

380

Acknowledgments

381

The research was funded by the Maj and Tor Nessling foundation and the Academy of

382

Finland, and Michigan the Center for Health Impacts in Agriculture. The authors thank the farms

383

for allowing the sampling and veterinarians Dr. Heli Simojoki, Mari Friman, Mari Niemi, Lotta

384

Wegelius and Prof. Mari Heinonen for the data on antibiotic use on farms. Dr. Jenni Hultman

385

and Dr. Christina Lyra are acknowledged for comments improving this article.


Page 16 of 35

Page 17 of 35


Supporting information S1

386 387 388

•

Figure S1. Venn diagrams showing the number of shared genes between samples.

389

•

Figure S2. Composition of positive assays in one or more samples grouped by

390

resistance mechanism and to antibiotic group the targeted gene confers resistance

391 392 393

•

Figure S3. Heat map showing relative abundances in samples taken from different

•

Figure S4. Fold change of genes from fresh manure (I) to stored manure (M),

farms

394

unfertilized soil (SB) to soil 2 weeks after fertilization and unfertilized soil (SB) to soil 6 weeks

395

after fertilization (S6WA)

396

•

Figure S5. Fold change of genes from ditch water (D) to ditch water 2 weeks after

397

soil fertilization (D2WA), ditch water (D) to ditch water 6 weeks after soil fertilization (D6WA)

398

and soil 2 weeks after fertilization (S2WA) to soil 6 weeks after fertilization (S6WA)

399

•

Table S1. Primer sets (assays) used in this study and their target classification.

400

•

Table S2. Results of statistical analysis for fold changes in compared samples.

401



402

References

403 404 405 406

1.

407

Health. Clin. Microbiol. Rev. 2011, 24, (4), 718-733.

408

2.

409

Animal Origin. Basic Clin. Pharmacol. Toxicol. 2005, 96, (4), 271-281.

410

3.

411

(5353), 996-997.

412

4.

413

Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Micro. 2010, 8,

414

(4), 251-259.

415

5.

416

Science 2008, 321, (5887), 365-367.

417

6.

418

environments. Curr. Opin. Biotechnol. 2008, 19, (3), 260-265.

419

7.

420

M.; Johnson-Rollings, A. S.; Jones, D. L.; Lee, N. M.; Otten, W.; Thomas, C. M.; Williams, A.

421

P., The role of the natural environment in the emergence of antibiotic resistance in Gram-

422

negative bacteria. Lancet Infect. Dis. 2013, 13, (2), 155-165.

423

8.

424

McAllister, T. A., Farm-to-fork characterization of Escherichia coli associated with feedlot cattle

425

with a known history of antimicrobial use. Int. J. Food Microbiol. 2010, 137, (1), 40-48.

Marshall, B. M.; Levy, S. B., Food Animals and Antimicrobials: Impacts on Human

Aarestrup, F. M., Veterinary Drug Usage and Antimicrobial Resistance in Bacteria of

Witte, W., Medical Consequences of Antibiotic Use in Agriculture. Science 1998, 279,

Allen, H. K.; Donato, J.; Wang, H. H.; Cloud-Hansen, K. A.; Davies, J.; Handelsman, J.,

Martínez, J. L., Antibiotics and Antibiotic Resistance Genes in Natural Environments.

Baquero, F.; Martínez, J.-L.; Cantón, R., Antibiotics and antibiotic resistance in water

Wellington, E. M. H.; Boxall, A. B. A.; Cross, P.; Feil, E. J.; Gaze, W. H.; Hawkey, P.

Alexander, T. W.; Inglis, G. D.; Yanke, L. J.; Topp, E.; Read, R. R.; Reuter, T.;


Page 18 of 35

Page 19 of 35


426

9.

427

A.; Navarro, F.; Alonso, M. P.; Bou, G.; Blanco, J.; Llagostera, M., Isolation and

428

Characterization of Potentially Pathogenic Antimicrobial-Resistant Escherichia coli Strains from

429

Chicken and Pig Farms in Spain. Appl. Environ. Microbiol. 2010, 76, (9), 2799-2805.

430

10.

431

J.; Donabedian, S.; Jensen, L. B.; Francia, M. V.; Baquero, F.; Peixe, L., Human and Swine

432

Hosts Share Vancomycin-Resistant Enterococcus faecium CC17 and CC5 and Enterococcus

433

faecalis CC2 Clonal Clusters Harboring Tn1546 on Indistinguishable Plasmids. J. Clin.

434

Microbiol. 2011, 49, (3), 925-931.

435

11.

436

Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and

437

Aquaculture Environments: Implication of Tn1721 in Dissemination of the Tetracycline

438

Resistance Determinant Tet A. Appl. Environ. Microbiol. 2000, 66, (9), 3883-3890.

439

12.

440

Biol. Rev. 2010, 74, (3), 417-433.

441

13.

442

Brown, H.; Davis, S.; Kay, P.; Boxall, A. B. A.; Wellington, E. M. H., Impacts of anthropogenic

443

activity on the ecology of class 1 integrons and integron-associated genes in the environment.

444

ISME J. 2011, 5, (8), 1253-1261.

445

14.

Mazel, D., Integrons: agents of bacterial evolution. Nat Rev Micro 2006, 4, (8), 608-620.

446

15.

Netherwood, T.; Bowden, R.; Harrison, P.; O’Donnell, A. G.; Parker, D. S.; Gilbert, H.

447

J., Gene Transfer in the Gastrointestinal Tract. Appl. Environ. Microbiol 1999, 65, (11), 5139-

448

5141.

449

16.

450

Maxwell, S.; Aminov, R. I., Fate and Transport of Antibiotic Residues and Antibiotic Resistance

Cortés, P.; Blanc, V.; Mora, A.; Dahbi, G.; Blanco, J. E.; Blanco, M.; López, C.; Andreu,

Freitas, A. R.; Coque, T. M.; Novais, C.; Hammerum, A. M.; Lester, C. H.; Zervos, M.

Rhodes, G.; Huys, G.; Swings, J.; McGann, P.; Hiney, M.; Smith, P.; Pickup, R. W.,

Davies, J.; Davies, D., Origins and Evolution of Antibiotic Resistance. Microbiol. Mol.

Gaze, W. H.; Zhang, L.; Abdouslam, N. A.; Hawkey, P. M.; Calvo-Bado, L.; Royle, J.;

Chee-Sanford, J. C.; Mackie, R. I.; Koike, S.; Krapac, I. G.; Lin, Y.-F.; Yannarell, A. C.;



451

Genes following Land Application of Manure Waste. J. Environ. Qual. 2009, 38, (3), 1086-

452

1108.

453

17.

454

microflora: environment. Int. J. Antimicrob. Agents 2000, 14, (4), 321-325.

455

18.

456

need to learn more about commensal flora if we are to better manage this particular window of

457

vulnerability to antibiotic resistance. ASM News 2003, 69, 601–607.

458

19.

459

responses. Nat. med. 2004, 10, (12 Suppl), S122-S129.

460

20.

461

the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J. 2007, 1,

462

(3), 191-203.

463

21.

464

Microbiol. 2010, 13, (5), 589-594.

465

22.

466

S. A.; Tiedje, J. M., Diverse and abundant antibiotic resistance genes in Chinese swine farms.

467

Proc. Natl. Acad. Sci. U.S.A. 2013, 110, (9), 3435-3440.

468

23.

469

T.; Esteban, J. E.; Currier, R. W.; Smith, K.; Thu, K. M.; McGeehin, M., Antimicrobial residues

470

in animal waste and water resources proximal to large-scale swine and poultry feeding

471

operations. Sci. Total Environ. 2002, 299, (1–3), 89-95.

472

24.

473

Antibiotic Resistance Gene Abundances in Archived Soils since 1940. Environ. Sci. Technol.

474

2010, 44, (2), 580-587.

Witte, W., Ecological impact of antibiotic use in animals on different complex

Andremont, A., Commensal flora may play key role in spreading antibiotic resistance we

Levy, S. B.; Marshall, B., Antibacterial resistance worldwide: causes, challenges and

Ghosh, S.; LaPara, T. M., The effects of subtherapeutic antibiotic use in farm animals on

Wright, G. D., Antibiotic resistance in the environment: a link to the clinic? Curr. Opin.

Zhu, Y.-G.; Johnson, T. A.; Su, J.-Q.; Qiao, M.; Guo, G.-X.; Stedtfeld, R. D.; Hashsham,

Campagnolo, E. R.; Johnson, K. R.; Karpati, A.; Rubin, C. S.; Kolpin, D. W.; Meyer, M.

Knapp, C. W.; Dolfing, J.; Ehlert, P. A. I.; Graham, D. W., Evidence of Increasing


Page 20 of 35

Page 21 of 35


475

25.

476

Kronberg, L.; Virta, M., Fertilizing with Animal Manure Disseminates Antibiotic Resistance

477

Genes to the Farm Environment. J. Environ. Qual. 2016, 45, (2), 488-493.

478

26.

479

resistomes. Nat. Rev. Micro. 2015, 13, (2), 116-123.

480

27.

481

W. J.; Stedtfeld, T. M.; Chai, B.; Cole, J. R.; Hashsham, S. A.; Tiedje, J. M.; Stanton, T. B., In-

482

feed antibiotic effects on the swine intestinal microbiome. Proc. Natl. Acad. Sci. U.S.A 2012,

483

109, (5), 1691-1696.

484

28.

485

Profiling of Antibiotic Resistance Genes in Urban Park Soils with Reclaimed Water Irrigation.

486

Environ. Sci. Technol. 2014, 48, (16), 9079-9085.

487

29.

488

Kim, D.; Lim, H. S.; Hashsham, S. A.; Tiedje, J. M.; Sul, W. J., Influence of Soil Characteristics

489

and Proximity to Antarctic Research Stations on Abundance of Antibiotic Resistance Genes in

490

Soils. Environ. Sci. Technol. 2016, 50, (23), 12621-12629.

491

30.

492

The Resistome of Farmed Fish Feces Contributes to the Enrichment of Antibiotic Resistance

493

Genes in Sediments below Baltic Sea Fish Farms. Front. Microbiol. 2017, 7, 2137.

494

31.

495

FunGene: the functional gene pipeline and repository. Front. Microbiol. 2013, 4, 291.

496

32.

497

Gulari, E.; Tiedje, J. M.; Hashsham, S. A., Development and Experimental Validation of a

498

Predictive Threshold Cycle Equation for Quantification of Virulence and Marker Genes by

Ruuskanen, M.; Muurinen, J.; Meierjohan, A.; Pärnänen, K.; Tamminen, M.; Lyra, C.;

Martinez, J. L.; Coque, T. M.; Baquero, F., What is a resistance gene? Ranking risk in

Looft, T.; Johnson, T. A.; Allen, H. K.; Bayles, D. O.; Alt, D. P.; Stedtfeld, R. D.; Sul,

Wang, F.-H.; Qiao, M.; Su, J.-Q.; Chen, Z.; Zhou, X.; Zhu, Y.-G., High Throughput

Wang, F.; Stedtfeld, R. D.; Kim, O.-S.; Chai, B.; Yang, L.; Stedtfeld, T. M.; Hong, S. G.;

Muziasari, W. I.; Pitkänen, L. K.; Sørum, H.; Stedtfeld, R. D.; Tiedje, J. M.; Virta, M.,

Fish, J. A.; Chai, B.; Wang, Q.; Sun, Y.; Brown, C. T.; Tiedje, J. M.; Cole, J. R.,

Stedtfeld, R. D.; Baushke, S. W.; Tourlousse, D. M.; Miller, S. M.; Stedtfeld, T. M.;



499

High-Throughput Nanoliter-Volume PCR on the OpenArray Platform. Appl. Environ. Microbiol.

500

2008, 74, (12), 3831-3838.

501

33.

502

for Statistical Computing,Vienna, Austria, 2015.

503

34.

504

Boston, MA, 2015, www.rstudio.org/ (accessed 30 Dec. 2016).

505

35.

506

2009.

507

36

508

T.;Maechler, M.; Magnusson, A.; Moeller, S.; Schwartz, M; Venables, B., gplots: Various R

509

Programming Tools for Plotting Data. 2016, R package version 3.0.1, https://CRAN.R-

510

project.org/package=gplots

511

37

512

Minchin, P.R.; O'Hara, R. B.;. Simpson G. L; Solymos, P.; Stevens, M.H.H.; Szoecs, E.;

513

Wagner, H., vegan: Community Ecology Package. 2016, R package version 2.4-1,

514

https://CRAN.R-project.org/package=vegan

515

38

516

Northwestern University, Evanston, Illinois, USA, 2016, R package version 1.6.9,

517

https://CRAN.R-project.org/package=psych.

518

39.

519

recommended alternative to Bonferroni-type adjustments in health studies. J. Clin. Epidemiol

520

2014, 67, (8), 850-857.

521

40. Bastian M., Heymann S., Jacomy M., Gephi: an open source software for exploring and

522

manipulating networks. International AAAI Conference on Weblogs and Social Media, 2009.

R Core Team, R: A Language and Environment for statistical computing. R Foundation

Rstudio Team, RStudio: Integrated development environment for R. RStudio, Inc.,

Wickham, H., ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York,

Warnes, G.R.; Bolker, B.; Bonebakker, L.; Gentleman, R.; Huber, W.; Liaw, A.; Lumley,

Oksanen, J.; Blanchet, F.G.; Friendly, M.; Kindt, R.; Legendre, P.; McGlinn, D.;

Revelle, W., psych: Procedures for Personality and Psychological Research,

Glickman, M. E.; Rao, S. R.; Schultz, M. R., False discovery rate control is a


Page 22 of 35

Page 23 of 35


523

41.

524

the class 1 integron-integrase gene as a proxy for anthropogenic pollution. ISME J. 2015, 9, (6),

525

1269-1279.

526

42.

527

countries in 2013. European Surveillance of Veterinary Antimicrobial Consumption 2015,

528

EMA/387934/2015.

529

http://www.ema.europa.eu/docs/en_GB/document_library/Report/2015/10/WC500195687.pdf

530

(accessed 19 Jan. 2017).

531

43.

532

Tetracycline Resistance in Agricultural Soils: Microcosm and Field Studies. Microb. Ecol. 2006,

533

51, (3), 267-276.

534

44.

535

FEMS Microbiol. Lett. 2003, 220, (1), 15-20.

536

45.

537

Tamminen, M.; Tiedje, J. M.; Virta, M., Aquaculture changes the profile of antibiotic resistance

538

and mobile genetic element associated genes in Baltic Sea sediments. FEMS Microbiol. Ecol.

539

2016, 92, (4), fiw052

540

46.

541

Micro 2006, 4, (8), 629-636.

542

47.

543

metagenomics reveals diverse β-lactamases in a remote Alaskan soil. ISME J 2008, 3, (2), 243-

544

251.

545

48.

546

antibiotic-resistant bacteria in soil following manure fertilization. Proc. Natl. Acad. Sci. U.S.A

547

2014, 111, (42), 15202-7.

Gillings, M. R.; Gaze, W. H.; Pruden, A.; Smalla, K.; Tiedje, J. M.; Zhu, Y.-G., Using

European Medicines Agency, Sales of veterinary antimicrobial agents in 26 EU/EEA

Schmitt, H.; Stoob, K.; Hamscher, G.; Smit, E.; Seinen, W., Tetracyclines and

Onan, L. J.; LaPara, T. M., Tylosin-resistant bacteria cultivated from agricultural soil.

Muziasari, W. I.; Pärnänen, K.; Johnson, T. A.; Lyra, C.; Karkman, A.; Stedtfeld, R. D.;

Piddock, L. J. V., Multidrug-resistance efflux pumps – not just for resistance. Nat Rev

Allen, H. K.; Moe, L. A.; Rodbumrer, J.; Gaarder, A.; Handelsman, J., Functional

Udikovic-Kolic, N.; Wichmann, F.; Broderick, N. A.; Handelsman, J., Bloom of resident



548

49.

549

Temporal changes of antibiotic-resistance genes and bacterial communities in two contrasting

550

soils treated with cattle manure. FEMS Microbiol Ecol 2016, 92, (2), fiv169

551

50.

552

S. V.; Daniels, J. B.; Wittum, T. E., Carbapenemase-producing Enterobacteriaceae recovered

553

from the environment of a swine farrow-to-finish operation in the United States. Antimicrob.

554

Agents Chemother. 2016, 61, (2), e01298-16.

555

51.

556

Smalla, K., Detection and characterization of broad-host-range plasmids in environmental

557

bacteria by PCR. Appl. Environ. Microbiol.1996, 62, (7), 2621-8.

558

52.

559

Independent, Tetracycline Resistance-Encoding Plasmid from a Dairy Enterococcus faecium

560

Isolate. Appl. Environ. Microbiol.2011, 77, (20), 7096-7103.

561

53.

562

a tet(M) Tetracycline Resistance Determinant Homologue in Clinical Isolates of Escherichia

563

coli. J. Bacteriol. 2006, 188, (20), 7151-7164.

564

54.

565

Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical Escherichia coli Strains

566

Isolated from Diverse Human and Animal Sources. Appl. Environ. Microbiol 2004, 70, (4),

567

2503-2507.

568

55.

569

unusual and diverse set of integrated gene transfer elements. Microbiol. Rev. 1995, 59, (4), 579-

570

590.

571

56.

572

mobile elements conferring antibiotic resistance. FEMS Microbiol. Rev. 2011, 35, (5), 856-871.

Hu, H.-W.; Han, X.-M.; Shi, X.-Z.; Wang, J.-T.; Han, L.-L.; Chen, D.; He, J.-Z.,

Mollenkopf, D. F.; Stull, J. W.; Mathys, D. A.; Bowman, A. S.; Feicht, S. M.; Grooters,

Götz, A.; Pukall, R.; Smit, E.; Tietze, E.; Prager, R.; Tschäpe, H.; van Elsas, J. D.;

Li, X.; Alvarez, V.; Harper, W. J.; Wang, H. H., Persistent, Toxin-Antitoxin System-

Jones, C. H.; Tuckman, M.; Murphy, E.; Bradford, P. A., Identification and Sequence of

Bryan, A.; Shapir, N.; Sadowsky, M. J., Frequency and Distribution of Tetracycline

Salyers, A. A.; Shoemaker, N. B.; Stevens, A. M.; Li, L. Y., Conjugative transposons: an

Roberts, A. P.; Mullany, P., Tn916-like genetic elements: a diverse group of modular


Page 24 of 35

Page 25 of 35


573

57.

574

Association with Different Transposons in the Antibiotic-Resistant Streptococcus pneumoniae.

575

Biomed Res Int. 2015 (2015), 836496.

576

58.

577

Elements in Tetracycline-Resistant Pneumococci and Correspondence between Tn1545 and

578

Tn6003. Antimicrob. Agents Chemother. 2008, 52, (4), 1285-1290.

579

59.

580

Cluster aadE–sat4–aphA-3 Disseminated among Multiresistant Isolates of Enterococcus

581

faecium. Antimicrob. Agents Chemother. 2001, 45, (11), 3267-3269.

582

60.

583

Trends Microbiol. 5, (10), 389-394.

584

61.

585

Skurray, R. A., The 3' conserved segment of integrons contains a gene associated with multidrug

586

resistance to antiseptics and disinfectants. Antimicrob. Agents Chemother. 1993, 37, (4), 761-

587

768.

588

62.

589

Class 1 Integrons among Salmonella Serotypes. Antimicrob. Agents Chemother. 2000, 44, (8),

590

2166-2169.

591

63.

592

determinants in Aeromonas spp. from rainbow trout farms in Australia. J. Fish Dis. 2011, 34,

593

(8), 589-599.

594

64.

595

comparison of bacteria from swine faeces and manure storage pits. Environ. Microbiol. 2003, 5,

596

(9), 737-745.

Korona-Glowniak, I.; Siwiec, R.; Malm, A., Resistance Determinants and Their

Cochetti, I.; Tili, E.; Mingoia, M.; Varaldo, P. E.; Montanari, M. P., erm(B)-Carrying

Werner, G.; Hildebrandt, B.; Witte, W., Aminoglycoside-Streptothricin Resistance Gene

Recchia, G. D.; Hall, R. M., Origins of the mobile gene cassettes found in integrons.

Paulsen, I. T.; Littlejohn, T. G.; Rådström, P.; Sundström, L.; Sköld, O.; Swedberg, G.;

Guerra, B.; Soto, S.; Cal, S.; Mendoza, M. C., Antimicrobial Resistance and Spread of

Ndi, O. L.; Barton, M. D., Incidence of class 1 integron and other antibiotic resistance

Cotta, M. A.; Whitehead, T. R.; Zeltwanger, R. L., Isolation, characterization and



597

65.

598

genes at cold temperatures: implications for winter storage of sludge and biosolids. Lett Appl.

599

Microbiol. 2014, 59, (6), 587-593.

600

66.

601

A Weitz, D.; Pitkänen, L. K.; Vigneault, F.; Virta, M. P.; Alm, E. J., Massively parallel

602

sequencing of single cells by epicPCR links functional genes with phylogenetic markers. ISME

603

J. 2016, 10, (2), 427-436.

604

67.

605

concentrations. Curr. Opin. Microbiol. 2006, 9, (5), 445-453.

606

68.

607

Antimicrob. Agents Chemother. 2012.

608

69.

609

dissemination of antibiotic resistance genes. Nature 2004, 427, (6969), 72-74.

610

70.

611

Gonzalez-Zorn, B.; Barbé, J.; Ploy, M.-C.; Mazel, D., The SOS Response Controls Integron

612

Recombination. Science 2009, 324, (5930), 1034.

613

71.

614

Induces Genetic Transformability in the Human Pathogen Streptococcus pneumoniae. Science

615

2006, 313, (5783), 89-92.

616

72.

617

Nat Rev Micro 2014, 12, (7), 465-478.

618

73.

619

Escherichia coli. Proc. Natl. Acad. Sci. U.S.A 1990, 87, (15), 5589-5593.

Miller, J. H.; Novak, J. T.; Knocke, W. R.; Pruden, A., Elevation of antibiotic resistance

Spencer, S. J.; Tamminen, M. V.; Preheim, S. P.; Guo, M. T.; Briggs, A. W.; Brito, I. L.;

Davies, J.; Spiegelman, G. B.; Yim, G., The world of subinhibitory antibiotic

Poole, K., Bacterial stress responses as determinants of antimicrobial resistance.

Beaber, J. W.; Hochhut, B.; Waldor, M. K., SOS response promotes horizontal

Guerin, É.; Cambray, G.; Sanchez-Alberola, N.; Campoy, S.; Erill, I.; Da Re, S.;

Prudhomme, M.; Attaiech, L.; Sanchez, G.; Martin, B.; Claverys, J.-P., Antibiotic Stress

Andersson, D. I.; Hughes, D., Microbiological effects of sublethal levels of antibiotics.

VanBogelen, R. A.; Neidhardt, F. C., Ribosomes as sensors of heat and cold shock in


Page 26 of 35

Page 27 of 35


620

74.

621

G., Bacterial phylogeny structures soil resistomes across habitats. Nature 2014, 509, (7502),

622

612-616.

Forsberg, K. J.; Patel, S.; Gibson, M. K.; Lauber, C. L.; Knight, R.; Fierer, N.; Dantas,

623



Page 28 of 35

Tables

624 625 626 627 628

Table 1. Farm codes, farm locations, animal numbers, the numbers of animals medicated with antibiotics and farming practices Farm code

Location (N/lat, E/lon)

Farm type

Number of animals

Number of animals medicated with Penicillins

Tetracycline

Fluoroqinolone

Tylosin

Manure storage

Soil texture and management

60°44'43'' Dairy farm

T1

180

4

2

1

0

Concrete silo

Clay loam No till

26°6'29''

60°34'10' T2

Clay

Dairy farm

240

Swine farm

1000

27

1

0

0

Lagoon No till

25°41'47''

60°47'43'' T3

233

0

0

12

Concrete silo

Sandy clay loam No till

25°13'51''

61°0'42'' Swine farm

T4

5000

141

10

0

24°22'26''

629 630


4

Concrete silo

Glacial till Tillage

Crop Grass (Festuca pratensis [Huds.] & Phleum pretense [L.]) Grass (Festuca pratensis [Huds.] & Phleum pretense [L.])

Rape (Brassica rapa [DC.])

Barley (Hordeum vulgare [L.])

Page 29 of 35

631 632


Table 2. Models used in permutational multivariate analysis. All presented R2 -values had significance at P

Influence of Manure Application on the Environmental Resistome

Recommend Documents