Bacteria That Make a Meal of Sulfonamide Antibiotics: Blind Spots and

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Critical Review

Bacteria that make a meal of sulfonamide antibiotics: blind spots and emerging opportunities Yu Deng, Bing Li, and Tong Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06026 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Environmental Science & Technology

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Bacteria that make a meal of sulfonamide antibiotics:

3

blind spots and emerging opportunities

4 Yu Deng1, Bing Li2 and Tong Zhang1

5 6 7

1

8

University of Hong Kong, Pokfulam Road, Hong Kong

9

2

10 11

Environmental Biotechnology Laboratory, Department of Civil Engineering, The

Division of Energy & Environment, Graduate School at Shenzhen, Tsinghua

University, Shenzhen 518055, China Corresponding author, +852-28578551; fax:+852-25595337; [email protected]

ACS Paragon Plus Environment


12

ABSTRACT

13 14

The release of sulfonamide antibiotics into the environment is alarming, because the

15

existence of these antibiotics in the environment may promote resistance in clinically

16

relevant microorganisms, which is a potential threat to the effectiveness of antibiotic

17

therapies. Controllable biodegradation processes are of particular significance for the

18

inexpensive yet effective restoration of sulfonamide-contaminated environments.

19

Cultivation-based techniques have already made great strides in successfully isolating

20

bacteria with promising sulfonamide degradation abilities, but the implementation of

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these isolates in bioremediation has been limited by unknown microbial diversity, vast

22

population responsiveness, and the impact of perturbations from open and complex

23

environments. Advances in DNA sequencing technologies and metagenomic analyses

24

are being used to complement the information derived from cultivation-based

25

procedures. In this review, we provide an overview of the growing understanding of

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isolated sulfonamide degraders and identify shortcomings of the prevalent literature in

27

this field. In addition, we propose a technical paradigm that integrates experimental

28

testing with metagenomic analysis to pave the way for improved understanding and

29

exploitation of these ecologically important isolates. Overall, this review aims to

30

outline how metagenomic studies of isolated sulfonamide degraders are being applied

31

for the advancement of bioremediation strategies for sulfonamide contamination.


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INTRODUCTION

33 34

Introduced as the earliest first-line antibiotics1, sulfonamides remain one of the most

35

purchased antibiotic classes currently on the market and are mainly used for the

36

intensification of food animal production in agriculture2. Recent survey data reported

37

that the sales of sulfonamides in 2011 accounted for nearly 11% of the total sales of

38

veterinary antibiotics in 25 European countries.3 As a result of their extensive use,

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sulfonamides are frequently detected in contamination sources and environmental

40

compartments (Figure 1). Three levels of contaminated wastewater can be

41

distinguished based on the detected concentrations of sulfonamide residues: (1)

42

wastewater derived from pharmaceutical production factories; (2) wastewater derived

43

from livestock farms, hospitals, and municipal sewer systems; and (3) discharged

44

effluent from wastewater treatment plants (WWTPs). Concentrations of sulfonamide

45

residues were found to be highest in pharmaceutical wastewater (up to 1340 µg/L4),

46

followed by the second and third types of wastewaters (Table 1). Extremely high

47

sulfonamide concentrations were also found in manure (up to 18 mg/kg5). When

48

investigated (Table S1), surface waters receiving wastewater from contaminated

49

WWTP effluents or livestock farms were seen to have higher sulfonamide

50

concentrations (max. concentration of 2.1±2.9 µg/L, number of detected datasets

51

(n)=4) than counterparts situated far from contamination sources (0.2±0.4 µg/L, n=10).

52

Moreover, a decreasing trend in sulfonamide concentrations was observed when the

53

sulfonamide residues were transported away from the sewage discharge point via

54

surface waters.6, 7 In addition to surface waters, quantitative survey data have also

55

indicated the spatial movement of sulfonamide residues from livestock wastewater

56

lagoons to hydraulically downgradient groundwater8, and a low-level yet continuous

57

transport of sulfonamide residues from manure to soil through common agricultural



58

manure fertilization practices5, 9. A more striking indication of the ubiquitous

59

occurrence of sulfonamide residues is the detection of these residues in drinking water

60

samples at maximal concentrations between 3 and 116 ng/L10-14. Of the reviewed

61

publications pertaining to sulfonamide contamination sources and the associated

62

environmental compartments (Table 1), the highest prevalence was observed for

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sulfamethoxazole (SMX, 92 positive samples out of 101 total sampled datasets),

64

followed by sulfamethazine (SMT, 40/101) and sulfadiazine (SDZ, 33/101).

65 66

Sulfonamides at such concentrations in the environment were found to be not acutely

67

toxic but mutagenic to most aquatic organisms tested (reviewed by García-Galán et

68

al.15). For example, the reported acute median effective or lethal concentration values

69

(E/LC50s) of SMX, which is the most frequently detected sulfonamide, for the marine

70

bacterium Vibrio fischeri (Microtox® test) ranged from 16.916 to 118.7 mg/L17.

71

However, Bengtsson-Palme et al.18 estimated that the no-effect concentration for

72

SMX-resistance selection was only 16 µg/L. The sulfonamide concentrations in

73

contamination hotspots (including wastewaters derived from pharmaceutical factories,

74

livestock farms and hospitals) could possibly exert a selective pressure on resistant

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pathogens, which can threaten therapeutic effectiveness19. To reduce the

76

environmental concentrations of sulfonamides, in addition to the optimization of the

77

use of sulfonamide antibiotics in human and animal treatments, we must prioritize the

78

development of remediation processes. Sulfonamide transformation in natural or

79

engineered environment involves both abiotic processes20-25 and biotic processes26-31.

80

Abiotic degradation such as photolysis and hydrolysis of the photo- and thermostable2

81

sulfonamides found at contaminated sites, is less likely to be significant than biotic

82

degradation. Upon the release of sulfonamides to wastewater and soil, which is a

83

common occurrence32, the

adsorption of sulfonamides by sludge or soil particles


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was negligible, as indicated by their low distribution coefficients (Kd values ranging

85

from 0.6 to 4.9)33 , while aerobic sulfonamide biodegradation is assumed to play a

86

critical role2, 34. The microbially mediated process is of significant research interests

87

since it can be implicated as a simple, inexpensive and environmentally friendly

88

remediation strategy. An array of bacteria that can aid in the environmental

89

remediation of sulfonamide-contaminated hotspot sites through biodegradation have

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been isolated and characterized using molecular and physiological techniques.

91

Additional experimental studies have recently started to reveal the genetic

92

determinants of sulfonamide degradation, until recently, the sole sulfonamide

93

catabolism gene, sadA, has been heterologously expressed and functionally validated

94

in Escherichia coli host cells35.

95 96

In this review, we highlight studies that shed light on the bacteria that are responsible for

97

sulfonamide biodegradation, and we present new considerations that must be addressed when

98

studying the factors affecting the degradation efficiencies of bacteria that specialize in

99

sulfonamide degradation. The integration of appropriate experimental testing with

100

metagenomics is discussed in the context of studying sulfonamide biodegradation in research

101

areas with different levels of microbial complexity, ranging from individual isolates and

102

closed and artificial systems, to open and complex environment matrices. Restrictions in the

103

translational implications of the proposed technical guide are also briefly discussed.

104 105

BACTERIAL PLAYERS IN SULFONAMIDE BIODEGRADATION

106 107

Sulfonamide-degrading bacteria can survive and thrive in the presence of sulfonamide

108

antibiotics. In addition to breaking down the mother compound, in some cases, these

109

degraders can subsist on sulfonamides as sole carbon sources. In this section, we first



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provide a framework that defines the concepts of sulfonamide resistance, tolerance,

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catabolism and subsistence in bacteria. Then, past attempts to

112

sulfonamide-degrading or sulfonamide-subsisting isolates are presented along with

113

the newly identified gene that initiate sulfonamide biodegradation. The risks

114

presented by sulfonamide resistant degraders in the environment are discussed at the

115

end of this section, as are possible approaches for the management of these risks.

identify

116 117

Sulfonamide resistance and tolerance in bacteria

118 119

Sulfonamide antibiotics can outcompete their close structural analog p-aminobenzoic

120

acid (PABA) on binding to a catalytic enzyme (i.e., dihydropteroate synthase (DHPS))

121

in the folate synthesis pathway, thereby inhibiting bacterial growth.36 Resistance

122

against sulfonamides emerged only six years after their introduction into the clinic in

123

19351. Resistance allows bacteria to sustain and thrive in the presence of antibiotics

124

and is usually evaluated by the minimal inhibitory concentration (MIC) of the

125

antibiotic, i.e., the minimum antibiotic concentration that prevents the net growth of a

126

bacterium.37 Bacteria typically resort to target-alteration strategy to resist

127

sulfonamides. This strategy exploits two mechanisms of preventing sulfonamides

128

from binding to DHPS, namely, chromosomal resistance resulting from mutations in

129

the DHPS gene (folP) and, more frequently, plasmid-borne resistance conferred by an

130

alternative DHPS gene (sul) whose enzyme product has a lower binding affinity for

131

sulfonamides than DHPS.36 On the other hand, the recently identified two-component

132

flavin-dependent

133

mechanism. This sadA gene-encoded enzyme has no sequence homology to known

134

sulfonamide resistance genes, and can inactivate sulfonamides in Escherichia coli by

135

previously undescribed oxidative mechanisms35.

monooxygenase

represent

a

novel


sulfonamide

resistance

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136 137

Different from resistance, tolerance to sulfonamide antibiotics was reported to be the

138

results of physiological adaptations38 in bacterial variants39. During a transient

139

antibiotic treatment, even at concentrations that are higher than the MIC37, tolerance

140

to antibiotics could enable the survival of these variants through a dormancy

141

mechanism40. It should be noted that tolerant variants are still antibiotic sensitive (as

142

reflected by the MIC value, which is the same as that of susceptible strains) and can

143

be killed with extended antibiotic treatment, and therefore, it has been suggested that

144

tolerance should be evaluated by the minimum duration for killing (MDK), i.e., the

145

minimum antibiotic treatment period that can kill a certain proportion of the bacteria

146

at different concentrations.37

147 148

Sulfonamide catabolism and subsistence by bacterial isolates

149 150

Bacteria capable of sulfonamide catabolism can evade sulfonamide-induced toxicity

151

while, counterintuitively, exhibiting the ability to degrade sulfonamides. Sulfonamide

152

catabolism by bacteria is thought to involve either (1) the action of common

153

resistance genes (sul) in tandem with sulfonamide catabolism genes that are unrelated

154

to the resistance mechanism, or (2) the sole action of sulfonamide catabolism genes

155

that provide resistance through degradation mechanisms (e.g. sadA gene). The first

156

sulfonamide-degrading bacterium was isolated almost ten years ago41, and a large

157

amount of follow-up studies isolated a set of bacteria that can degrade or transform

158

disparate classes of sulfonamides (including sulfamethoxazole, SMX; sulfamethazine,

159

SMT; sulfadiazine, SDZ; sulfadimethoxine, SDM; sulfapyridine, SPY; and

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sulfathiazole, STZ) at a concentration range of 600 ng/L42 to 152 mg/L43 (Table 2).

161

Phylogenetic profiling of the isolated bacteria revealed a diverse range of species in



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the phyla Proteobacteria (81%, 39 out of 48) and Actinobacteria (19%) (Figure 2). Of

163

the 17 genera represented, Burkholderia constituted most (23%) of the species

164

isolated, followed by Pseudomonas (15%), Ralstonia (10%), and Microbacterium

165

(10%). The capabilities of these isolates to catabolize sulfonamides were variable, as

166

were their original habitats (Figure 3a). Activated sludge and soil were the main

167

habitats from which 34 out of the 48 sulfonamide-degrading bacteria were isolated.

168 169

Analyses of the degradation products revealed that the heterocyclic moieties of

170

sulfonamides were typically accumulated as a stable metabolite in a few

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representative isolates, independent of the specific substrates or conditions provided

172

(Figure 3b). Though the final extent of sulfonamide mineralization in pure cultures

173

was generally limited by the degradation abilities of the isolates, the heterocyclic

174

moieties formed by sulfonamide breakdown, such as 3-amino-5-methylisoxazole

175

(3A5MI, SMX heterocyclic moiety), were shown to have lower potential

176

environmental impacts due to the loss of the antibacterial action of sulfonamides42, 44.

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For example, during SMX degradation by the strain Microbacterium sp. BR1, 3A5M

178

was detached from SMX, releasing 4-iminoquinone and sulfur dioxide simultaneously

179

(C10H11N3O3S (SMX) + OH- → C6H5NO (benzoquinone-imine) + SO2 + C4H5N2O-

180

(3A5MI-) + 2H+ + 2e-).45 Comparative analyses on proteomic patterns of sulfonamide

181

presence-absence conditions during BR1’s cultivation, together with sequencing of

182

partially

183

monooxygenase was the catabolic enzyme that initiated the breakdown of SMX via a

184

strategy that pairs ipso-hydroxylation with subsequent fragmentation of the mother

185

compound (Figure 3b), and this reaction mechanism allows the maintenance of the

186

molecular skeleton of the leaving groups35. In silico genomic analyses further found

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sadA homologues in other four sulfonamide degraders (including Arthrobacter sp. D2

purified

protein

fraction

indicated

that


the

sadA

gene-encoded

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and D4, Microbacterium sp. C448 and Microbacterium sp. strain SDZm4) which were

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isolated from different continents35. Despite a few studies45,

190

obtained evidences on genes that initiated the sulfonamide degradation, the newly

191

identified sadA gene was the only sequence that was functionally validated.

46

have previously

192 193

Of the 17 bacterial genera that can degrade or transform sulfonamides, with the

194

exception of the genus Alcaligenes47, 48, 16 genera have demonstrated the ability to

195

use sulfonamides as the sole carbon source. The capacity of a bacterium to deploy

196

antibiotics for energy and biomass growth was defined as antibiotic subsistence41, 49.

197

Bacteria subsisting on sulfonamides are often isolated by plating serial dilutions of an

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inoculum (usually an enriched culture of naturally occurring microbial communities)

199

on sulfonamide-containing mineral salts medium (MSM) solidified with agar. Notably,

200

the growth of a colony on a sulfonamide-MSM agar plate cannot be taken as direct

201

evidence of its degradation ability. Degradation ability should be confirmed in liquid

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medium, in which cell growth coupled with the disappearance of the sulfonamide can

203

be observed. It is crucial to emphasize that the sulfonamide-MSM should contain

204

certain sulfonamides as sole carbon and energy sources, and alternative carbon

205

sources should be avoided. Initial attempts to obtain sulfonamide-subsisting bacteria

206

have to the isolation of 18 soil bacteria with the ability to grow on either

207

sulfamethizole (SMZ) or sulfisoxazole (SSZ) as a sole carbon source41 (Table 2 and

208

Figure 3a). In Particular, two seawater strains50, Escherichia sp. HS21 and

209

Acinetobacter sp. HS51, as well as Microbacterium sp. BR151 and Achromobacter

210

denitrificans PR143 were shown to be capable of subsisting on a variety of

211

sulfonamides

212 213

Limitations of cultivation-based studies



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Current studies on isolates subsisting on sulfonamide have been focused on isolating

216

these degraders, characterizing the extent of degradation and the metabolites produces,

217

and evaluating the effects of the sulfonamide concentration42, 52, the presence of

218

additional carbon substrates (e.g., glucose53, tryptone52, sodium-acetate54, succinate43),

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the carbon to nitrogen ratio in the medium54, pH and temperature52,

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degradation efficiencies of these degraders. A few studies have experimentally proven

221

the carbon mineralization of the sulfonamide aniline moiety to CO2 and biomass using

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isotope-labeled sulfonamides43, 45, 51, 57. For example, during degradation experiments

223

with Microbacterium lacus SDZm4 using

224

moiety, 56% and 16% of the applied radioactivity were detected in the CO2 and

225

biomass produced, respectively; however, in parallel experiments using pyrimidine

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ring-labeled SDZ, all of the applied radioactivity was identified in the dead-end

227

metabolite, 2-aminopyrimide (2-AP).57 These experiments demonstrated that it was

228

the limited ability of strain SDZm4 to assimilate carbon from the SDZ pyrimidine

229

moiety that prevented complete SDZ mineralization. One study43 showed a general

230

improvement in the SMX degradation efficiency of Achromobacter denitrificans PR1

231

with either the addition of a mixture of 18 kinds of amino acids alone or the addition

232

of a combination of these 18 kinds of amino acids with 14 kinds of vitamins and

233

nitrogenous bases. Zhang et al.47 reported that 100 mg/L exogenous vitamin C,

234

vitamin B6 or oxidized glutathione significantly enhanced the SMX degradation by

235

Alcaligenes faecalis CGMCC 1.767, while no enhancements were observed when

236

vitamin B2 and vitamin B12 were added. However, only Ricken et al.35specifically

237

identified FMNH2 as an indispensable cofactor in the SMX degradation by

238

Microbacterium sp. strain BR1. Micronutrients involved in the sulfonamide

239

subsistence of isolated degraders require further research.

14

55, 56

on the

C-labeled SDZ, labeled at its aniline


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Even though the sulfonamide degradation abilities of the characterized isolates were

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promising in laboratory microcosms, the results from the application of these

243

sulfonamide-degrading strains under conditions similar to those in the field situations

244

were often disppointmenting58, 59. The uncertainty in the extrapolation of the results from

245

laboratory degradation to field processes mainly stems from two related issues: the

246

interactions within the microbial communities; and the correlations between community

247

members and environmental variables. To date, mechanistic insights into the above issues are

248

very limited, partially due to the difficulties in using traditional cultivation-based techniques

249

to decipher the roles of key sulfonamide degraders in complex microbial communities.

250

Metagenomics, a technique that applies shotgun sequencing to genomic fragments extracted

251

from a microbial community, has been demonstrated to be a cultivation-independent and

252

high-resolution method for probing microbial structure and functions in both natural60-62 and

253

engineered63-65 communities.

254 255

APPLYING METAGENOMICS TO SULFONAMIDE DEGRADERS

256 257

The genetic determinants of sulfonamide degradation are key factors that must be

258

studied to understand and exploit sulfonamide degraders. In this section, two

259

strategies (comparative genomics and functional metagenomics) for the fast screening

260

of catabolism genes in sulfonamide-degrading isolates or various environmental

261

microorganisms are discussed. In addition, the use of metagenomic profiling to

262

monitor population responsiveness to sulfonamide exposure within a simplified

263

microbial community is also demonstrated, with a focus on addressing issues

264

associated with experimental design (Figure 4). The application of metagenomic

265

analyses to the study of the correlations between degrader effectiveness and



266

environmental variables is also briefly discussed.

267 268

Screening of sulfonamide catabolism genes

269 270

The newly identified sulfonamide catabolism gene (sadA) was the only sequence that

271

was functionally validated in heterologous cells, and its homologues were only found

272

in sulfonamide degraders affiliating with the phylum Actinobacteria. However, since

273

around 81% of the known sulfonamide degraders were belonging to the phylum

274

Proteobacteria, the diversity of the sulfonamide catabolism genes is still needed to be

275

discovered. With increasingly available genomic sequences of sulfonamide degraders

276

on public database like NCBI, genomic mining and physiological characterization of

277

sulfonamide-degrading isolates and their phylogenetic relatives can provide

278

information about the genomic basis of sulfonamide biodegradation. An illustrative

279

implementation of this strategy has been performed using three Dehalococcoides

280

strains to correlate their genomic contents with their disparate dechlorination

281

profiles66. The observed physiological differences among those strains were found to

282

be dictated by the presence of distinct reductive dehalogenase-encoding genes with

283

specific chlorinated ethane functions. Though highly informative, comparative

284

genomic analyses are unable to directly measure gene function. Tiered experimental

285

designs encompassing both genome-physiology relationships and transcriptomic

286

analyses may facilitate more robust functional annotations of identified gene

287

candidates.

288 289

Another technique that has been demonstrated to be useful in the discovery of novel

290

catabolic genes is functional metagenomics. This technique can facilitate the retrieval

291

of genetic information from complex environmental matrices for various phenotypes


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without culturing efforts67. The utility of functional metagenomics has been

293

exemplified by recent applications of this method for the functional elucidation of

294

novel enzymes such as hydrolase68, carbon-fixation enzymes69 and bilirubin-oxidizing

295

enzymes70. Briefly, this technique paires cloning and functional selection of

296

environmental DNA fragments with metagenomic sequencing71. Notably, for the gene

297

candidates discovered using the above mentioned approaches, functional validation in

298

a host is needed to confirm sulfonamide-degrading activities of these candidates.

299

Since the facile hosts commonly used for functional expression are gram-negative

300

bacteria (such as Escherichia coli), functional expression experiments probably

301

preclude the discovery of gram-positive bacteria-specific genes.

302 303

Identifying the roles of individual isolates

304 305

As might be expected given the cultivation limitations, the roles of isolates from

306

sulfonamide-degrading consortia have not been identified. There is a lack of

307

information linking the identities of these isolates to their activities in artificial or

308

natural habitats. Additionally, the mechanisms by which isolated degraders establish

309

and maintain their membership within a community, especially the as-yet-uncultured

310

members that may be involve in sulfonamide degradation, remain unknown.

311

Incorporation of the genomic data of sequenced sulfonamide-degrading isolates and

312

metagenomic studies of sulfonamide-degrading communities can complement and

313

expand the information derived cultivated bacteria.

314 315

A current highlight of metagenomic analysis is genome-resolved metagenomics,

316

which uses binning algorithms to extract genome sequences of individual species

317

from metagenomic datasets of environmental samples72-74 to evaluate community



318

membership. If the genomes of a certain sulfonamide-degrading isolate and a

319

reconstructed population bin are identical to each other (as reflected by identical

320

average amino acid or nucleotide identities; online calculation available at

321

http://enve-omics.ce.gatech.edu75), we can confidently assign the bin genome to the

322

matched isolate. In contrast to molecular analyses such as fluorescence in situ

323

hybridization (FISH), real-time quantitative PCR (RT-PCR) and DNA stable-isotope

324

probing (SIP), this explicitly proactive approach is independent of sample availability.

325

The growing publicly available metagenomic-data resources for environmental

326

samples (web-based platform includes IMG/ER, NCBI-SRA and MG-RAST)

327

currently provide a framework within which the identification and surveillance of

328

sulfonamide-degrading isolates can be carried out in diverse microbial ecosystems. It

329

should be noted that the presence of a matched sulfonamide-degrading isolate in an

330

environmental sample cannot be viewed as evidence that sulfonamide biodegradation

331

is occurring in situ; ecological validation of field activity is still required.

332 333

Tracking microbial community responses to sulfonamide exposure

334 335

Monitoring of microbial community responses to sulfonamide exposure, including

336

determining community compositions and tracking changes in the abundance of

337

various members present in the community76, can provide information for the

338

prediction of microbial interaction processes (such as competition for nutrients) that

339

underpin steady biodegradation. Several studies have investigated the community

340

changes in response to sulfonamide exposure, often via 16S rRNA gene-based

341

molecular methods (Table S2). The community responses measured in these studies

342

were variable, probably due to the heterogeneity of the initial inoculum communities

343

or due to different sulfonamide additions and carbon source amendments (including


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344

manure77, spent mushroom compost78, 79, glucose80, yeast extract81, sodium acetate82-84,

345

and other assimilable carbon sources85; see Table S2 for treatment specifications).

346 347

Nevertheless, even with this variable background in these studies, reduced microbial

348

diversitywas commonly observed as a shared feature. In fact, when sulfonamides

349

were supplied as the sole carbon source, the decrease in community diversity could be

350

accounted for by the selective force for bacteria specializing in sulfonamide

351

degradation, such as Achromobacter and Pseudomonas spp86. In particular, there were

352

a few studies that examined the effects of sulfonamides as the sole carbon source on

353

microbial community dynamics. The time frames for these enrichment studies ranged

354

from days to months. Even with short term enrichment, substantial changes in the

355

composition of the initial inoculum community were observed. One study that

356

performed sulfanilamide enrichment using sulfonamide-contaminated lake water as

357

inoculum revealed that the dominant Burkholderia spp. were rapidly succeeded by

358

Bacilli and Flavobacteriia members over a period of 48 days87. These enrichment

359

processes indicated the abilities of the microorganisms to assimilate sulfonamides.

360

Enrichment culturing of naturally occurring microbial communities with devised

361

growth conditions is an applicable strategy for the identification of novel

362

sulfonamide-degrading isolates. For instance, in a recent application of enrichment

363

culturing for the specific purpose of isolating SDZ degraders, activated sludge from a

364

municipal wastewater treatment plant was used to inoculate a defined mineral salts

365

medium containing SDZ (50 mg/L) as the sole carbon source46. After 10-month

366

incubation, when the SDZ was extensively and stably mineralized (~85% TOC

367

reduction within 72 h), small volumes of the enriched culture were spread onto SDZ

368

mineral-salts agar. Single colonies that grew on SDZ were selected, further purified,

369

identified and characterized by appropriate physiological procedures and phylogenetic



370

classifications. Two Arthrobacter strains capable of subsisting on SDZ were obtained,

371

revealing an additional, previously unappreciated ecological significance for

372

Arthrobacter. Sulfonamide degraders are relatively straightforward to isolate by using

373

enrichment culturing strategies. However, the ecological roles of these isolates in the

374

enriched communities are difficult to identify based on phylogenetic analyses.

375 376

Moving towards degradation in the open environment

377 378

The remarkable propensity of microorganisms to environmental perturbations is

379

critical for enrichment culturing processes; however, this propensity can be a major

380

impediment for biodegradation studies in mesocosm and field conditions. Occurring

381

as responses to nutrient availability or variations in physicochemical parameters,

382

quantitative or qualitative alterations in the interactions within microbial populations

383

(such as competition, predation and cooperation) and the relevant degradation

384

reactions are to be expected. A recent example is a soil bioremediation study using the

385

SMT-degrading strain C44859: The inoculation of a native soil containing 1 mg/kg

386

SMT with strain C448 failed to increase SMT mineralization rate, indicating the low

387

activity and poor survival of the inoculated strain C448 in the environment to which it

388

was exposed.

389 390

Associations inferred from community dynamics can help determine the keystone

391

populations of distinct ecological relevance and the responsiveness of these

392

populations to nutritional substrates and physiological conditions, which in turn can

393

guide the design of cultivation media so that ecologically significant microorganisms

394

can be isolated, and the relevant functional genes from these microorganisms can be

395

activated under laboratory conditions. With respect to sulfonamide biodegradation,


Page 16 of 46

Page 17 of 46


396

the dynamics of sulfonamide-degrading bacteria were mainly followed by

397

amplicon-based sequencing using the 16S rRNA gene (Table S2). However,

398

phylogenetic analysis based on the 16S rRNA gene was insufficient for the

399

determination of strain-level variations, necessitating the implementation of other

400

analytical methods under the umbrella of shotgun metagenomic sequencing (reviewed

401

in the references88,

402

microbiomes. Sulfonamide biodegradation studies can take a nested approach

403

incorporating microbial association networks and whole-community ordination to

404

identify consistent patterns of successful biodegradation at the community level.

405

Microbial association networks are a powerful tool for modeling and visualizing the

406

statistical co-occurrence of clusters of keystone species90, 91 that cannot be examined

407

directly. Network features include positive and negative co-occurrence. Positive

408

co-occurrence may suggest symbiosis or shared preferences for environmental

409

conditions, and negative co-occurrence may arise due to competition or distinct

410

preferences for mutually exclusive environmental conditions. Visualization of these

411

network features has often been performed via analysis platforms such as Gephi92 and

412

Cytoscape93. On the other hand, whole-community ordination analyses, such as

413

non-metric multidimensional scaling and canonical correspondence analysis, can

414

simplify the complexities associated with environmental variability by visualizing the

415

data in two or three dimensions94, thereby linking community structures with

416

environmental

417

biodegradation.

89

) for the analysis of community dynamics of diverse

determinants

that

are

conducive

to

enhanced

sulfonamide

418 419

Limitations in the translational implications of the proposed technical guide

420 421

There are three important questions which can help to improve our understanding on



Page 18 of 46

422

biodegradation, including identifying the catabolic genes that initiates the

423

biodegradation, deciphering the complexity of pollutant-degrading communities, as

424

well as increasing impetuses for the inquiry of the ecological role of isolated

425

degraders. And all these important steps towards progress in biodegradation studies

426

are articulated in Figure 4, thus this figure can illustrate how metagenomics

427

approaches are deployed in studies concerning other pollutant biodegradation.

428

However, metagenomic approaches have a fundamental limitation, that is, the

429

degradation activity of an isolate or a microbial community under a certain condition

430

cannot be measured. Thus, in order to fully describe the relationship between genetic

431

controls and degradation processes, additional multi-omic data, such as the levels of

432

RNA (i.e., transcriptomics), are needed, preferably integrated with appropriate

433

experimental designs.

434 435

OUTLOOK

436 437

Extensive survey of the widespread occurrence of sulfonamides in diverse

438

environment matrices has brought attention to sulfonamide contamination. As the

439

amount of residual sulfonamides that enter the environment can be anticipated to

440

increase, the importance of in situ remediation of sulfonamide contaminant is

441

becoming

442

sulfonamide-contaminated water are focused on chemical methods such as advanced

443

oxidation processes95-97. Though highly efficient in removing the mother compounds,

444

the high costs of the application of these methods and the production of highly toxic

445

daughter products during the transformation processes98,

446

methods less desirable for in situ sulfonamide remediation. By contrast, sulfonamide

447

biodegradation is more likely to be the predominant mechanism for field remediation.

apparent.

Preliminary

studies

aimed


at

the

99

remediation

of

make these chemical

Page 19 of 46


448 449

Given

that

sulfonamide

subsistence

represents

alternative

sulfonamide

450

resistance-mechanism, a question that remains unanswered is whether degraders that

451

subsist on sulfonamides are the “bugs” that contribute to the increasingly elevated

452

levels of antibiotic resistance in certain environment matrices. This paradox is mainly

453

associated with the specialized catabolic niche in these degraders, which enables them

454

to be tolerant to these toxins and confers on them a selection advantage over other

455

environmental microorganisms in the presence of sulfonamides. A recent study that

456

examined resistance mechanisms of natural benthic biofilm communities exposed to

457

high sulfonamide concentrations proposed that sulfonamide biodegradation is an

458

adaptive resistance strategy for survival100. In addition, the distribution of bacteria that

459

subsist on sulfonamides in the open environment would probably result in the

460

detoxification of the surrounding microenvironment, which would be beneficial to

461

other susceptible pathogens sharing that microenvironment. In our opinion, due to the

462

above mentioned ecological concerns, control strategies (e.g. membrane separation

463

and subsequent incineration) used for bioremediation of contaminated sites using with

464

sulfonamide-subsisting bacteria have the potential to prevent the dissemination of

465

antibiotic resistance carried by these degraders. As a pragmatic measure that combats

466

antibiotic resistance by reducing the direct exposure of environmental microbial

467

populations to sulfonamides, in situ remediation of hotspots is essential. When taking

468

steps to further develop sulfonamide bioremediation strategies, it is important to

469

acknowledge the risks involved with certain degraders that have the ability to subsist

470

on antibiotics.

471 472

Before drawing wider conclusions by interpreting the degradation abilities measured

473

for sulfonamide-degrading isolates or enriched consortia, however, it is important to



474

address certain considerations; for example, it is important to know whether the

475

laboratory results can be extrapolated to open environmental conditions. Identifying

476

ecologically significant species and understanding their interactions with other

477

community members or environmental variables hold the key to the empirical

478

development of sulfonamide bioremediation. Complementary and convergent

479

information derived from cultivation-based procedures and sequencing-based

480

techniques will surely make these questions increasingly addressable.

481 482

SUPPLEMENTARY INFORMATION

483 484

This information is available free of charge via the Internet at http://pubs.acs.org/.

485

This file contains two supplementary tables (Table S1 and S2).

486 487

AUTHOR INFORMATION

488 489 490

Corresponding Author Phone: +852-28578551; Fax:+852-25595337; E-mail: [email protected]

491

Notes

492

The authors declare no competing financial interest.

493 494

ACKNOWLEDGMENTS

495 496

This work was funded by the Theme-based Research Scheme (T21-711/16-R). Yu

497

Deng would like to thank The University of Hong Kong for the postgraduate

498

studentship.


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Cultures. PloS one 2016, 11, (10), e0165013.



1046 1047 1048

Table of Contents (TOC) Art


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Page 37 of 46


1049

Table and Figure Legends

1050 1051

Table 1.

contamination sources and environmental compartments

1052 1053

Maximum concentrations of the most frequently detected sulfonamides in

Table 2.

An overview of reported sulfonamide-degrading isolates

Figure 1.

Boxplot of sulfonamide occurrence in contamination sources and

1054 1055

environmental compartments.

1056 1057

Figure 2.

reported sulfonamide-degrading bacterial isolates.

1058 1059

Figure 3.

1062

An overview of bacterial isolates that subsist on sulfonamides (a) and their main degradation pathways (b).

1060 1061

A phylogenetic tree based on 16S rRNA gene sequences covering all

Figure 4.

Model for the integration of experimental testing and metagenomic analysis

1063



1064 1065

Page 38 of 46

Table 1. Maximum concentrations of the most frequently detected sulfonamides in contamination sources and environmental compartments Contamination source Pharmaceutical wastewater (2.05-1340 µg/L) Livestock wastewater (0.39-25.4 µg/L) Hospital wastewater (0.028-27.8 µg/L) WWTP influent (0.034-7.91 µg/L) WWTP effluent (0.015-9.46 µg/L) Manure (140-18000 µg/kg) Activated Sludge (0.04-665 µg/kg) Environmental compartment Surface water (0.01-12 µg/L) Groundwater (0.001-0.113 µg/L) Drinking water (0.003-0.116 µg/L) Soil (0.37-760.1 µg/L) Total sampled datasets

Detection

Sulfonamide

Max concentration

5/5

Sulfamethazine

3.41-139.7 µg/L

3/5

Sulfadiazine

2.05-697.4 µg/L

2/5

Sulfamethoxazole

24.8-1340 µg/L

6/6

Sulfamethazine

0.8-25.4 µg/L

4/6

Sulfathiazole

0.4-10.57 µg/L

3/6

Sulfamethoxazole

0.44-8.84 µg/L

12/12

Sulfamethoxazole

0.2-27.8 µg/L

3/12

Sulfadiazine

0.3-2.33 µg/L

2/12

Sulfamethazine

0.028-1.7 µg/L

14/14

Sulfamethoxazole

0.36-7.91 µg/L

105, 109, 111,

3/14

Sulfadiazine

0.094-5.15 µg/L

112, 114,

3/14

Sulfapyridine

0.15-3.27 µg/L

119-126

13/13

Sulfamethoxazole

0.09-9.46 µg/L

111,

4/13

Sulfadiazine

0.019-4.18 µg/L

117, 119-127

4/13

Sulfapyridine

0.18-0.446 µg/L

5/6

Sulfamethoxazole

840-18000 µg/kg

3/6

Sulfadiazine

800-1980 µg/kg

2/6

Sulfamerazine

140-4590 µg/kg

10/10

Sulfamethoxazole

0.11-665 µg/kg

5,

7/10

Sulfapyridine

13.3-296.4 µg/kg

133-139

6/10

Sulfadiazine

5.99-112.3 µg/kg

Detection

Sulfonamide

Max concentration

Referenc

13/13

Sulfamethoxazole

0.036-4.3 µg/L

4,

10/13

Sulfamethazine

0.065-6.19 µg/L

8/13

Sulfadiazine

0.013-2.3 µg/L

10/12

Sulfamethoxazole

0.009-1.11 µg/L

7/12

Sulfamethazine

0.013-0.6 µg/L

4/12

Sulfamerazine

0.05-0.744 µg/L

4/4

Sulfamethoxazole

0.03-0.116 µg/L

2/4

Sulfadimethoxine

0.011-0.092 µg/L

2/4

Sulfamethizole

0.09-0.098 µg/L

6/6

Sulfamethoxazole

0.9-671.5 µg/kg

5, 106, 129, 131,

4/6

Sulfamethazine

0.66-74 µg/kg

135, 159, 160

3/6

Sulfadiazine

4.26-760.1 µg/kg

101

Total references


Reference 4, 101-104

8, 101, 105-108

4, 101, 109-118

112,

117,

114,

5, 128-132

124,

6,

7,

125,

105,

140-148

8, 126, 149-158

10-14

70

Page 39 of 46


1068

Table2. An overview of reported sulfonamide-degrading isolates. aFor the first eighteen strains in the table41, positive biomass growth (at least 108cells/ml) was observed when the strains were supplied with sulfonamides as the sole carbon source; the concentration changes of sulfonamide were not recorded. bDuring the degradation process, the total cell number increased; the final biomass was not recorded. cFor strains BR1, BR2,

1069 1070

BR3, HB1 and HB251, sulfonamide removal was recorded as the extent of mineralization of SMX to CO2. dRate refers to the removal extent which was normalized to biomass and degradation time. MSM, mineral salts medium; MBR, lab-scale membrane bioreactor; RT, room

1071 1072

temperature; PBS, phosphate-buffered saline; ASBR, aerobic sequence batch reactor; NaAc, sodium acetate; – indicates that values are not available.

1066 1067

1073 Organism Burkholderia ginsengisoli Burkholderia ginsengisoli Serratia sp Methylobacterium sp Haemophilus sp Ralstonia pickettii

Strain

Habitat

Slfm-S1P-M1LLLSSL-1

Soil

Slfm-S1P-M1LLLSSL-2 Slfm-S1T-M1LLLSSL-1 Slfm-S1T-M1LLLSSL-2 Slfm-S1T-M1LLLSSL-3 Slfm-S2N-M1LLLSSL-1

Soil Soil Soil Soil Soil

Biodegradation conditions MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C

Biomass

Removala

Time

>108 cells/ml

–

30 d

8

–

30 d

8

–

30 d

8

–

30 d

8

–

30 d

8

–

30 d

8

>10 cells/ml >10 cells/ml >10 cells/ml >10 cells/ml >10 cells/ml

Ralstonia pickettii

Slfm-S2N-M1LLLSSL-2

Soil

MSM+1000 ppm SMZ, 26°C

>10 cells/ml

–

30 d

Ralstonia pickettii

Slfm-S2N-M1LLLSSL-3

Soil


>108 cells/ml

–

30 d

8

Burkholderia phenazinium

Slfm-S2R-M1LLLSSL-1

Soil


>10 cells/ml

–

30 d

Burkholderia phenazinium

Slfm-S2R-M1LLLSSL-2

Soil


>108 cells/ml

Burkholderia phenazinium Burkholderia sediminicola Burkholderia sediminicola

Slfm-S2R-M1LLLSSL-3 Slfm-S2T-M1LLLSSL-1 Slfm-S2T-M1LLLSSL-2

Soil Soil Soil

MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C


–

30 d

8

–

30 d

8

–

30 d

8

–

30 d

>10 cells/ml >10 cells/ml >10 cells/ml

Ref

41


Burkholderia sediminicola Burkholderia ginsengisoli Burkholderia ginsengisoli Burkholderia ginsengisoli

Slfm-S2T-M1LLLSSL-3 Slfm-S3F-M1LLLSSL-1 Slfm-S3F-M1LLLSSL-2 Slfm-S3F-M1LLLSSL-3

Serratia marcescens

Slfs-S3F-M1LLLSSL-3

Rhodococcus rhodochrous

ATCC 13808

Rhodococcus equi

ATCC 13557

Soil Soil Soil Soil Soil Purchased

Purchased


Microbacterium sp.

S-3

BR1

ASBR

MBR

>108 cells/ml

–

30 d

8

–

30 d

8

–

30 d

8

–

30 d

8

>10 cells/ml

–

30 d

MSM+32 ppm SMX+3 g/L glucose, 26°C

–

20%

36 d

MSM+43 ppm SMZ+3 g/L glucose, 26°C

–

14%

12 d

MSM+6 ppm SMX+0.5 g/L glucose, 26°C

OD540=1.36

29%

5d

MSM+6 ppm SMX, 26°C

OD540=0.64

15%

5d

Growing cellb

33.4%

2d

PBS+25 ppm SMX, RT45

100%

2.5 h

PBS+25 ppm SDZ, RT

100%

2h

100%

2h

PBS+31 ppm SDM, RT

100%

5h

PBS+28 ppm SMT, RT

100%

21 h

MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SMZ, 26°C MSM+1000 ppm SSZ, 26°C

MSM+5 ppm SMT+17 g/L glucose +10 g/L Achromobacter sp

Page 40 of 46

peptone + 3 g/L beef extract, 30°C

PBS+27 ppm SMZ, RT

>10 cells/ml >10 cells/ml >10 cells/ml

OD595=0.50


Growing cell

40%c

6.5h

BR3


Growing cell

44%

6.5h

Ralstonia sp

HB1


Growing cell

24%

6.5h

Ralstonia sp

HB2


Growing cell

44%

6.5h

Escherichia sp

HS21

66%

2d

45%

2d

Rhodococcus sp

BR2

Achromobacter sp

MBR

Seawater

MSM+10 ppm SPY, 28°C MSM+10 ppm STZ, 28°C


107 CFU/mL

161

53

55

51

50

Page 41 of 46


Acinetobacter sp

HS51

Seawater

MSM+10 ppm SPY, 28°C

72%

2d

67%

2d

10 CFU/mL

30%

2d

MSM+10 ppm SDZ, RT

–

100%

21 d

10% Mueller-Hinton broth+10 ppm SDZ, RT

–

100%

4.2 d

–

100%

–

MSM+10 ppm STZ, 28°C Pseudomonas sp

DX7

Microbacterium lacus

SDZm4

Seawater Soil

MSM+10 ppm SDX +2-8 g/L tryptone , 30°C

107 CFU/mL 7

Microbacterium sp

C448

Soil

MSM+ 50 ppm SMT

Brevundimonas sp

SMXB12

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

Rated (mg/L/d), 2.5/1.7/1.0

Microbacterium sp

SMXB24

AS


Rate (mg/L/d), 2.5/1.25/1.25

Microbacterium sp

SMX348

AS


Rate (mg/L/d), 2.5/1.7/1.25

Pseudomonas sp

SMX321

AS


Rate (mg/L/d), 2.5/2.5/1.7

Pseudomonas sp

SMX330

AS


Rate (mg/L/d), 2.5/1.7/1.25

Pseudomonas sp

SMX331

AS


Rate (mg/L/d), 2.5/1.7/1.25

Pseudomonas sp

SMX344

AS


Rate (mg/L/d), 2.5/1.7/1.25

Pseudomonas sp

SMX345

AS


Rate (mg/L/d), 2.5/1.25/1.25

Variovorax sp

SMX332

AS


Rate (mg/L/d), 2.5/1.7/1.25

Pseudomonas psychrophila

HA-4

AS


Protein=0.1 g/L

34.3%

8d

MSM+152 ppm SMX

OD600=1.00

100%

14 d

MSM+152 ppm SMX +590 ppm succinate

OD600=1.00

100%

1.8 d

MSM+25 ppm SDZ

OD600=1.00

98%

2.3 d

MSM+31 ppm SDM

OD600=1.00

48%

2.3 d

MSM+28 ppm SMT

OD600=1.00

98%

2.3 d

MSM+25 pm SPY

OD600=1.00

100%

2.3 d

Achromobacter denitrificans

PR1

AS


52

57

162

54

56

43


MSM+26 pm STZ

Page 42 of 46

OD600=1.00

47%

2.3 d

OD540=0.20

94%

16 h

OD595=0.30

100%

53 h

MSM+50 ppm SMX+ 1 g/L NaAc+1 g/L Alcaligenes faecalis

CGMCC 1.767

Arthrobacter sp

D2

Purchased AS

glucose+100 ppm Vitamin C, 30°C MSM+50 ppm SMX, 37°C

47

46


OD595=0.30

99%

11 d

Manure

MSM+5 ppm SDZ+0.04% yeast extract, 30°C

OD600=~0.8

50%

12 d

SDZ-W2-SJ40

AS


OD600=~1.0

55%

12 d

SDZ-3S-SCL47

Sediment


OD600=~1.1

60%

12 d

Arthrobacter sp

D4

Paracoccus sp

DZ-PM2-BSH30

Methylobacterium sp Kribbella sp

AS

1074


163

Page 43 of 46


10.0

1000

15000

0.9

600

20 7.5 0.6

10000

0.3

5000

200

0

0

0

400

5.0 500

10

2.5

0

1075 1076 1077 1078

0

Pharmaceutical wastewater

Figure 1.

0

Livestock Hospital WWTP wastewater wastewater influent

WWTP effluent

Surface water

Groundwater

Drinking water

Manure

Soil

Sewage sludge

Boxplot of sulfonamide occurrence in contamination sources and environmental compartments. The presented data summarized the maximal concentrations of 11types of frequently encountered sulfonamides from 70 publications. The three most frequently detected sulfonamides are listed in Table 1. Details of the concentration values (both maximum and average) are included in Table S1.



Sl Sl fm-S f Sl m-S 3Ff Sl m-S 2R- M1L M fm 2 Slf -S R-M 1LL LLS m- 2R S Slf S1 -M 1LL LSS L-1 L 1 m Slf -S3 P-M1 LLL SSL L-3 m F Slf -S3 -M1 LLLSSSL -2 F -1 m L β-Proteobacteria Slf -S1P -M1L LLS SL-2 mS L M S2 Slf 1L LSS L-3 N m L -M L Slfm -S2N- 1LL LSSL -2 L M -1 Slfm S2N-M 1LLL SSL-3 S 1 Slfm S2T-M LLLS SL-2 S L1LL -S 2 T LSS 1 Slfm -M1 L Rals -S2T-M LLLSS -3 1LL L tonia L SSL -2 sp. H Rals to 1 B2 Vario nia s vorax p . HB sp. S 1 M X 332 Achro mob Achrom acter sp. PR Pseudom obacter 1 onas sp. sp. BR3 Pseudomon DX7 as psychrop

Ou

t-g r

ou p

Page 44 of 46

in g

Ni tro sp

ira

hila HA-4

Pseudomonas sp.

γ-Proteobacteria

SMX321 Pseudomonas sp. SMX3 30 Pseudomonas sp. SMX331 Pseudomonas sp. SMX345 Pseudomonas sp. SMX344 L-3 Slfm-S1T-M1LLLSS s-S3F-M1LLLSSL-3 Slf

HS51 HS21 aceticus cter sp. cter calco Acinetoba Enteroba 1LLLSSL-1

Tree scale: 0.01

1T-M L-2 Slfm-S LLLSS J40 1T-M1 2-S Slfm-S p. SDZ-W 30 s 2 -BSH 48 rium BR 3 12 bacte -PM2 p. α-Proteobacteria Methylo as sp. SMXuBs sp. SDZ sp. SMXXB24 ss u 7 c m 4 on SM iu 48 occ C L c oc cter m sp. p. C4 1 undim Parac Brev s R r oba iu S - S do Mic bacter erium sp. B m4 Z-3 Rho t D Z o c r m S Mic icroba cteriu us SD 4 p. a M as l a c p. D 2 rob ell Mic terium cter s p. D ribb K rs ac ba rob rthro acte Mi c A ob thr r Actinobacteria A

Sulfonamide-degrading isolates (n=48)

1079 1080 1081 1082 1083 1084 1085

Figure 2.

A phylogenetic tree based on 16S rRNA gene sequences covering all reported sulfonamide-degrading bacterial isolates. Phylogeny in this tree corresponds to 1000 bootstrap replicates. The tree has been rooted with the out-grouping 16S rRNA gene sequence from Nitrospira sp (GenBank accession number AJ224042).

.


Page 45 of 46


1086



1087 1088

Figure 3.

Page 46 of 46

Overview of bacterial isolates that subsist on sulfonamides (a) and their main degradation pathways (b). Bacterial isolates capable of utilizing specific sulfonamides as sole carbon sources are colored based on their original habitat. Detailed information for the individual strains is included in Table 2.

1089 1090 1091

Sulfonamide as sole C source Assembly and annotation

Amplify and sequencing

Extrapolation of labscale results Roles of sulfonamidedegrading isolates

Individual

Community complex level

Populations

1092 1093

1094 1095 1096

Figure 4.

Model for the integration of experimental testing and metagenomic analysis.

1097 1098


Bacteria That Make a Meal of Sulfonamide Antibiotics: Blind Spots and

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