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

Mar 2, 2018 - When taking steps to further develop sulfonamide bioremediation strategies, it is important to acknowledge the risks involved with certa...
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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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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]

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ABSTRACT

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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

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inexpensive yet effective restoration of sulfonamide-contaminated environments.

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

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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

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

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environments. Advances in DNA sequencing technologies and metagenomic analyses

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

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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

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

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

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

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outline how metagenomic studies of isolated sulfonamide degraders are being applied

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for the advancement of bioremediation strategies for sulfonamide contamination.

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INTRODUCTION

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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WWTP effluents or livestock farms were seen to have higher sulfonamide

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

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

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

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sulfonamide residues were transported away from the sewage discharge point via

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

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indicated the spatial movement of sulfonamide residues from livestock wastewater

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

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transport of sulfonamide residues from manure to soil through common agricultural

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

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

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

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publications pertaining to sulfonamide contamination sources and the associated

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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),

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

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

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

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

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

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

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

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

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contamination hotspots (including wastewaters derived from pharmaceutical factories,

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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

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

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

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development of remediation processes. Sulfonamide transformation in natural or

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

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

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

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

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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

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

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

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

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

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remediation of sulfonamide-contaminated hotspot sites through biodegradation have

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been isolated and characterized using molecular and physiological techniques.

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Additional experimental studies have recently started to reveal the genetic

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determinants of sulfonamide degradation, until recently, the sole sulfonamide

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

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in Escherichia coli host cells35.

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

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

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

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sulfonamide degradation. The integration of appropriate experimental testing with

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

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

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

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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

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

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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

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

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the newly identified gene that initiate sulfonamide biodegradation. The risks

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

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

identify

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Sulfonamide resistance and tolerance in bacteria

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Sulfonamide antibiotics can outcompete their close structural analog p-aminobenzoic

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

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

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

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

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

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

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

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sulfonamides. This strategy exploits two mechanisms of preventing sulfonamides

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

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

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

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

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flavin-dependent

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

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

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previously undescribed oxidative mechanisms35.

monooxygenase

represent

a

novel

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sulfonamide

resistance

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

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results of physiological adaptations38 in bacterial variants39. During a transient

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

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

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

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

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

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

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

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at different concentrations.37

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Sulfonamide catabolism and subsistence by bacterial isolates

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Bacteria capable of sulfonamide catabolism can evade sulfonamide-induced toxicity

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

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

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

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

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

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

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

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

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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).

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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

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

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

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

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

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

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Analyses of the degradation products revealed that the heterocyclic moieties of

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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

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

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

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

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

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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

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

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

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

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

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partially

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

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

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

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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

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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,

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

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

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have previously

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

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

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

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

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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)

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

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

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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

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

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

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

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

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

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

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

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

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sulfonamides

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Limitations of cultivation-based studies

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

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

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

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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

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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

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with Microbacterium lacus SDZm4 using

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

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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

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

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

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

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improvement in the SMX degradation efficiency of Achromobacter denitrificans PR1

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

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

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

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vitamin B6 or oxidized glutathione significantly enhanced the SMX degradation by

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Alcaligenes faecalis CGMCC 1.767, while no enhancements were observed when

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

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identified FMNH2 as an indispensable cofactor in the SMX degradation by

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Microbacterium sp. strain BR1. Micronutrients involved in the sulfonamide

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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

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

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

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

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interactions within the microbial communities; and the correlations between community

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

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

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

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

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

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

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engineered63-65 communities.

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APPLYING METAGENOMICS TO SULFONAMIDE DEGRADERS

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

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

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

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of catabolism genes in sulfonamide-degrading isolates or various environmental

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

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monitor population responsiveness to sulfonamide exposure within a simplified

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

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

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analyses to the study of the correlations between degrader effectiveness and

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environmental variables is also briefly discussed.

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Screening of sulfonamide catabolism genes

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

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

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

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

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

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discovered. With increasingly available genomic sequences of sulfonamide degraders

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

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sulfonamide-degrading isolates and their phylogenetic relatives can provide

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information about the genomic basis of sulfonamide biodegradation. An illustrative

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implementation of this strategy has been performed using three Dehalococcoides

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strains to correlate their genomic contents with their disparate dechlorination

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

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

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specific chlorinated ethane functions. Though highly informative, comparative

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

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designs encompassing both genome-physiology relationships and transcriptomic

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analyses may facilitate more robust functional annotations of identified gene

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candidates.

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

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

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of genetic information from complex environmental matrices for various phenotypes

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without culturing efforts67. The utility of functional metagenomics has been

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

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

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

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

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candidates discovered using the above mentioned approaches, functional validation in

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

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

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

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

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Identifying the roles of individual isolates

304 305

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

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

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

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

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

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

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

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metagenomic studies of sulfonamide-degrading communities can complement and

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expand the information derived cultivated bacteria.

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

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which uses binning algorithms to extract genome sequences of individual species

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from metagenomic datasets of environmental samples72-74 to evaluate community

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

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

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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

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

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

324

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

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The growing publicly available metagenomic-data resources for environmental

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

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currently provide a framework within which the identification and surveillance of

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

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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

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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

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

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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.

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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

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

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

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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

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

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

369

identified and characterized by appropriate physiological procedures and phylogenetic

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

371

revealing an additional, previously unappreciated ecological significance for

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Arthrobacter. Sulfonamide degraders are relatively straightforward to isolate by using

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

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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,

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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

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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

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the

99

remediation

of

make these chemical

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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

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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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Table of Contents (TOC) Art

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Environmental Science & Technology

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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

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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

ACS Paragon Plus Environment

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

Environmental Science & Technology

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

MSM+1000 ppm SMZ, 26°C

>108 cells/ml



30 d

8

Burkholderia phenazinium

Slfm-S2R-M1LLLSSL-1

Soil

MSM+1000 ppm SMZ, 26°C

>10 cells/ml



30 d

Burkholderia phenazinium

Slfm-S2R-M1LLLSSL-2

Soil

MSM+1000 ppm SMZ, 26°C

>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

ACS Paragon Plus Environment



30 d

8



30 d

8



30 d

8



30 d

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

Ref

41

Environmental Science & Technology

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

MSM+1000 ppm SMZ, 26°C

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

MSM+127 ppm SMX, 28°C

Growing cell

40%c

6.5h

BR3

MSM+127 ppm SMX, 28°C

Growing cell

44%

6.5h

Ralstonia sp

HB1

MSM+127 ppm SMX, 28°C

Growing cell

24%

6.5h

Ralstonia sp

HB2

MSM+127 ppm SMX, 28°C

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

ACS Paragon Plus Environment

107 CFU/mL

161

53

55

51

50

Page 41 of 46

Environmental Science & Technology

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

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Microbacterium sp

SMX348

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas sp

SMX321

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas sp

SMX330

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas sp

SMX331

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas sp

SMX344

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas sp

SMX345

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Variovorax sp

SMX332

AS

10 ppm SMX in R2A/MSM+NaAc/MSM

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

Pseudomonas psychrophila

HA-4

AS

MSM+100 ppm SMX, 10°C

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

ACS Paragon Plus Environment

52

57

162

54

56

43

Environmental Science & Technology

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

MSM+50 ppm SMX, 37°C

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

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

OD600=~1.0

55%

12 d

SDZ-3S-SCL47

Sediment

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

OD600=~1.1

60%

12 d

Arthrobacter sp

D4

Paracoccus sp

DZ-PM2-BSH30

Methylobacterium sp Kribbella sp

AS

1074

ACS Paragon Plus Environment

163

Page 43 of 46

Environmental Science & Technology

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.

ACS Paragon Plus Environment

Environmental Science & Technology

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).

.

ACS Paragon Plus Environment

Page 45 of 46

Environmental Science & Technology

1086

ACS Paragon Plus Environment

Environmental Science & Technology

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

ACS Paragon Plus Environment