ipso-Hydroxylation and Subsequent Fragmentation: a Novel MicrobialStrategy To Eliminate Sulfonamide Antibiotics

Benjamin Ricken,a Philippe F. X. Corvini,a,b Danuta Cichocka,a Martina Parisi,a Markus Lenz,a,c Dominik Wyss,a

Paula M. Martínez-Lavanchy,d Jochen A. Müller,d Patrick Shahgaldian,e Ludovico G. Tulli,e Hans-Peter E. Kohler,f Boris A. Kolvenbacha

Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerlanda; State Key Laboratory ofPollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Chinab; Sub-Department of Environmental Technology, WageningenUniversity, Wageningen, The Netherlandsc; Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research—UFZ, Leipzig, Germanyd;Institute for Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerlande; Eawag, SwissFederal Institute of Aquatic Science and Technology, Department of Environmental Microbiology, Dübendorf, Switzerlandf

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in theenvironment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabo-lites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experi-ments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent frag-mentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl groupresulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly trans-formed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfon-amide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hy-droxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same orin a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to thesulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.

Sulfonamides are widely used as antibiotics, antidiabetics, di-uretics, antivirals, and anticancer agents (1–4), and thus, large

amounts of these compounds enter the environment every year(5, 6). Contamination with sulfonamides poses environmentalconcern due to the potential development and dissemination ofantibiotic resistances (7). Despite their ubiquity, their microbialmetabolism and ultimate fate in the environment thereof arepoorly understood.

Several studies showed that sulfamethoxazole (SMX) (Fig. 1a),an important representative of sulfonamide compounds, under-goes partial degradation in wastewater treatment plants underaerobic and anaerobic conditions (8–11). We recently demon-strated that Microbacterium sp. strain BR1, a Gram-positive bac-terium isolated from a membrane bioreactor treating effluentcontaminated by several pharmaceuticals, was capable of miner-alizing the 14C-labeled aniline moiety of SMX when the latter wassupplied as the sole carbon source (12). This was the first unam-biguous indication that sulfonamides are subject to growth-linkedmetabolism in microorganisms.

To our knowledge, Hartig (13) was the first to identify theaminated heteroaromatic side moieties of the sulfonamides SMXand sulfadimethoxine as stable metabolites after biodegradationwith activated sludge. This result was recently confirmed by twogroups that were able to isolate Microbacterium strains with theability to degrade the sulfonamides sulfamethazine (SMZ) (14)and sulfadiazine (SDZ) (15). Additionally, both groups identifiedthe aminated heteroaromatic side moieties of the sulfonamide as astable metabolite after the degradation of the parent compound.Although these stable metabolites were identified, the initial attackof the sulfonamide and the further degradation pathway of theaniline path remained unclear.

Before those findings were reported, only few sulfonamide me-

tabolites (i.e., hydroxylated, acetylated, and glycosylated, etc.)were identified, which were not further degraded (16–18).

Most commercial sulfonamides are para-substituted aromaticamines. As hydroxyl groups, amino moieties are ortho- and para-directing activators in electrophilic aromatic substitutions, andortho- and para-substituents of phenolic compounds can be de-tached by ipso-substitution (19–28). Such biologically mediatedipso-substitutions proceed according to an addition eliminationmechanism whereby an electrophilic oxygen species attacks thearomatic ring at the carbon atom bearing the substituent (ipso-position), leading to the formation of a transient hydroxylatedcyclohexadienone intermediate. Subsequently, the substituent iseliminated as an anion (type I ipso-substitution) or as a cation(type II ipso-substitution), and para-quinone or quinol, respec-tively, arises as the product (23, 28). The type of elimination de-pends on whether the ring-substituent bond electrons in the cyc-lohexadienone derivative remain with the substituent or with thering moiety. The reaction, regardless of type, is affected by elec-tronic and steric properties of both the substrate and the attackingagent (29), and the full range of biochemically catalyzed ipso-sub-stitutions remains as yet unexplored.

Here, we show that biologically mediated ipso-substitution

Received 21 March 2013 Accepted 1 July 2013

Published ahead of print 8 July 2013

Address correspondence to Philippe F. X. Corvini, philippe.corvini@fhnw.ch.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00911-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00911-13

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leads to a novel bacterial degradation pathway for SMX and sev-eral other sulfonamides. From the data, we postulate that the sul-fonamides are initially hydroxylated at the ipso-position, formingN-substituted 1-hydroxy-4-imino-cyclohexa-2,5-diene-1-sul-fonamide intermediates, which immediately undergo fragmenta-tion to 4-iminoquinone, sulfur dioxide, and the aminated substit-uent. We discuss the implications of this pathway with regard toboth the environmental fate of sulfonamides and the developmentof resistance to sulfonamide antibiotics.

MATERIALS AND METHODSAcclimatization of Microbacterium. Microbacterium cells were acclima-tized to SMX by incubating the cells in 25% (vol/vol) Standard I medium(Merck, Grogg Chemie, Stettlen-Deisswil, Switzerland) with 0.5 mMSMX. The cultures were incubated on a rotary shaker (Multitron; InforsHT, Bottmingen, Switzerland) at 130 rpm at 28°C until the optical densityat 600 nm (OD600) reached 1.2 (after approximately 40 h). Cells used formineralization or degradation experiments were washed by centrifuga-tion at 4,500 � g at 4°C for 15 min (5804R; Eppendorf, Basel, Switzer-land). The supernatant was discarded, and the cells were suspended in themedium required for the subsequent experiment. This washing step wasrepeated at least three times.

Sulfonamide degradation assay. The sulfonamide degradation stud-ies were carried out with SMX, sulfadiazine (SDZ), sulfamethazine, sulfa-methizole, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide,and asulam (Fig. 1). The initial concentration of the sulfonamides was 0.1mM. A Microbacterium sp. strain BR1 cell suspension was diluted withphosphate-buffered saline (PBS) to an OD600 of 0.5, and all experimentswere carried out in triplicates. Ten milliliters of the cultures was incubatedin 50-ml reaction tubes with screw caps on a rotary shaker at 230 rpm at

room temperature (RT). Samples were taken every 30 min for 6 h andfinally after 21 h. Abiotic controls consisting of pure PBS and the corre-sponding sulfonamides were also analyzed in triplicates. The sulfon-amides sulfanilamide and 4-amino-N-cyclohexylbenzenesulfonamide(Fig. 1) were incubated at an OD600 of 7 as in the experiments describedabove, as an OD600 of 0.5 did not lead to detectable degradation. Fromthese batches, samples were taken after 0 and 1 h of incubation. In addi-tion, a control experiment with cells and SMX as a substrate was carriedout to verify the metabolic activity of the Microbacterium sp. strain BR1batch under the same conditions. After centrifugation of the samples,supernatants were analyzed by high-performance liquid chromatography(HPLC) to monitor the concentrations of the parent compound as well asthe corresponding degradation product, except for methylcarbamate (Fig.1n), which is expected to result from asulam (Fig. 1m) degradation. Therespective rates for degraded sulfonamides were determined in the lineardegradation range over at least 4 data points (2 h of incubation) and withan r2 of �0.98.

Growth assay using SMX as a carbon source. The growth of Micro-bacterium sp. strain BR1 was tested in a separate experiment. Cultureswere grown in 250-ml Erlenmeyer flasks filled with 100 ml of MMO me-dium (45) amended with yeast extract (0.5 mg liter�1) and vitamins (0.5ml liter�1) (46). SMX (0.5 mM) was added as an electron donor andcarbon source. A total of 0.05% of a culture (OD600 � 1) grown on Stan-dard I medium served as the inoculum. The cells were washed twice andresuspended in MMO medium as described above, before adding them tothe minimal medium. Cells with SMX, treatments inoculated with auto-claved inoculum, and treatments inoculated with cells but without SMXwere set up in triplicate. Cultures were incubated at 28°C on a rotaryshaking incubator (130 rpm). One milliliter of sample was centrifuged for30 min at 16,000 � g at 4°C, the supernatant was discarded, and the pelletwas stored at �80°C until DNA extraction was performed. The growth of

FIG 1 Structures of the sulfonamides tested for degradation by resting cells of Microbacterium and the stable transformation products formed from them. (a)Sulfamethoxazole; (b) 3-amino-5-methylisoxazole; (c) sulfadiazine; (d) 2-aminopyrimidine; (e) sulfadimethoxine; (f) 2,6-dimethoxy-4-pyrimidinamine; (g)sulfamethazine; (h) 2,6-dimethyl-4-pyrimidinamine; (i) sulfamethizole; (j) 5-methyl-1,3,4-thiadiazol-2-amine; (k) 4-amino-N-phenylbenzenesulfonamide; (l)aniline; (m) asulam; (n) methylcarbamate; (o) 4-amino-N-cyclohexylbenzenesulfonamide; (p) sulfanilamide.

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strain BR1 was measured by quantitative PCR (qPCR), as described in thesupplemental material.

4-Aminophenol degradation. In the degradation assays, the initial4-aminophenol concentration was 75 �M, and the OD600 of Microbacte-rium sp. strain BR1 cells was 7.0. Samples were taken for 4-aminophenolanalysis at 0, 7.5, 15, 30, 45, and 60 min after the start of incubation. Thesamples were directly mixed with ice-cold methanol (20% [vol/vol] finalconcentration) and stored in an ice bath containing water and ethanol inthe dark to stop both biotic and abiotic transformation of 4-aminophenol.All degradation experiments were set up in triplicates. The samples werethen centrifuged at 32,000 � g at 4°C for 5 min. 4-Aminophenol wasdetected in the supernatants by a colorimetric method adapted frommethods described previously by Van Bocxlaer et al. (30). One hundredmicroliters of Na2HPO4 (0.5 M in H2O [pH 12]) was mixed with 10 �lMnCl2 (1 mM in H2O) and 10 �l resorcinol (48 mM in H2O) before 100�l of the sample was added. After a 5-min reaction time, the absorptionwas measured at 550 nm on a Synergy 2 multimode microplate reader(Biotek, Luzern, Switzerland). Due to the abiotic oxidation of 4-amino-phenol, the starting concentration of the abiotic sample was measureddirectly after setting up the experiment. For the calculation of the4-aminophenol concentration from the absorption value, a standard wasfreshly prepared.

Measurements of oxygen consumption rates. Oxygen consumptionrate (OCR) measurements were performed by using an XF96 extracellularflux analyzer (Seahorse Bioscience, USA) based on fluorimetric O2 detec-tion. This system allows the measurement of 96 different oxygen-consum-ing reactions in parallel and the separated injection of 4 different sub-strates (ports A to D on the sensor cartridge). This system was proven to besuitable for assays of cultured cells (31) as well as oxygen-consumingenzymes (32). The hydration of the CFA96 sensor cartridge was carriedout overnight with 200 �l calibration solution per well. Just before thecalibration of the sensor cartridge, port A was loaded with 25 �l 50 mMPBS (pH 7), and port B was loaded with 25 �l of freshly prepared 400 �Msubstrate solutions in PBS (final reaction mixture concentration, 50 �M).The reaction microplate was loaded with 150 �l acclimatized and nonac-climatized Microbacterium. sp. strain BR1 cells (final OD600 of 2) for thebiotic assays or with PBS for abiotic controls. Every setup was carried outin quadruplicates at a constant temperature of 32°C. The protocol for themeasurements was as follows: mixing for 30 s, measurement for 20 min,injection of port A, mixing for 30 s, measurement for 10 min, injection ofport B, mixing for 30 s, and measurement for 4 h. The oxygen consump-tion rate was calculated in the linear range (r2 � 0.99) of oxygen consump-tion after injection of the substrate.

Cell disruption. For the 18O experiment, a frozen Microbacteriumstock with an OD600 of 6.7 in PBS was thawed at RT, and 1.5 mg ml�1

lysozyme (100,000 units mg�1; Fluka, Buchs, Switzerland) was added.The mixture was incubated on a rotary shaker (Swip; Edmund Bühler,Hechingen, Germany) at RT for 1 h. After centrifugation at 15,000 � g at4°C for 15 min, the supernatant was discarded, and the cell pellet wassuspended to the initial volume by adding ice-cold PBS. The cells weredisrupted with the OmniLyse HL kit (ClaremontBio Solutions, Upland,CA, USA). The OmniLyse bead chamber was prewashed with PBS inadvance, as described in the OmniLyse protocol, and each lysozyme-treated cell aliquot was disrupted separately by withdrawing and infusingit 10� through the bead chamber. The disrupted-cell suspension wasfinally centrifuged at 16,000 � g at 4°C for 10 min. The supernatants werepooled and used for the cell extract degradation experiments.

For all other experiments with cell extracts carried out on larger scales,frozen Microbacterium stocks with an OD600 of 30 were thawed andtreated with lysozyme as described above. After centrifugation at 15,000 �g at 4°C for 15 min, the cells were disrupted by sonication (80% ampli-tude, 0.5 cycle, for 20 min) in an ice bath containing water and ethanol at�3°C, after adding 10% (wt/wt) glass beads to the cell suspension. Thesuspension was then centrifuged at 60,000 � g at 4°C for 20 min. Theprotein content of the crude cell extract was measured with the Pierce

bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Olten,Switzerland).

Cell extract degradation assays. The cell extract degradation assayswere carried out with the undiluted crude cell extract in duplicates. SMXwas added in PBS to a final concentration of 100 �M. Cofactor stocks wereprepared freshly in PBS and added to the assay mixtures at a final concen-tration of 1 mM (NADH and NADPH, respectively). Samples were taken,and the proteins contained within were precipitated by adding 25% (vol/vol) 1 M HCl to the sample. The mixture was incubated for 15 min at RTbefore centrifugation at 16,000 � g at 4°C for 10 min. The supernatant wasanalyzed by HPLC.

For the cofactor dependence evaluation and the assays to determinesulfite formation from SMX in cell extracts, 300 �l of the sample wastransferred into ultrafiltration tubes (Amicon Ultra 10K device; Milli-pore, Germany) and centrifuged at 14,000 � g at 4°C for 15 min toretain proteins. The flowthrough was subjected to HPLC and ion chro-matography.

Incubations of cell extracts under an 18O2 atmosphere. Twenty-mil-liliter headspace gas chromatography-mass spectrometry (GC-MS) vials(Agilent Technologies, Germany) sealed with butyl-rubber stoppers wereflushed with nitrogen. Subsequently, 4 ml of 18O2 (isotopic purity, 97%;Sigma-Aldrich, Switzerland) was drawn by syringe from a vessel held up-side down under water before being filled with 18O2. The oxygen wasquickly injected into the vials after inserting a second needle for pressureequalization. Subsequently, a mixture of SMX and cell extract containing1.2 mg protein ml�1, prepared as described above, was added to the vialswithout cofactors; finally, 50 �l of aqueous NADH solution was added toa final concentration of 1 mM to start the reaction, while the final concen-tration of SMX was 0.1 mM in a 1-ml total volume. After 30 min ofincubation, the vials were opened, and 500 �l of the reaction mixture wassubjected to ultrafiltration in 0.5-ml centrifugal filters (Amicon Ultra 10Kdevice; Millipore, Germany) and centrifuged at 14,000 � g at 4°C for 15min to retain proteins. Filtrates were then transferred into HPLC vials forliquid chromatography-mass spectrometry (LC-MS) analysis.

RESULTSGrowth of Microbacterium sp. strain BR1 on mineral mediumwith sulfamethoxazole as the sole carbon source and identifica-tion of 3A5MI as a dead-end metabolite. Microbacterium sp.strain BR1 was able to grow on 0.5 mM (126.5 mg liter�1) SMX asthe principal source of carbon and energy when supplied withvitamins and trace amounts (0.5 mg liter�1) of yeast extract (seeFig. S1 in the supplemental material). Growth was determined byreal-time PCR quantifying the 16S rRNA gene copy number. Toidentify SMX degradation metabolites in growing cultures, we an-alyzed the supernatants by means of a high-performance liquidchromatograph coupled to a diode array detector (HPLC-DAD)and a high-performance liquid chromatograph coupled to a massspectrometer (LC-MS). We detected a UV-absorbing metabolite(measurable absorbance below 260 nm) that was absent in con-trols. The metabolite was identified as 3-amino-methylisoxazole(3A5MI) (Fig. 1b) by comparison of retention times, UV-visible(UV-Vis) absorption spectra, and electron spray ionization (ESI)mass spectra (see Fig. S2 in the supplemental material) with thecommercially available 3A5MI standard. Further experimentsperformed with resting cells of Microbacterium sp. strain BR1 re-vealed that the decrease in the concentration of SMX coincidedwell with the increase in the concentration of 3A5MI (see Fig. 2cand Fig. S3 in the supplemental material for HPLC-DAD chro-matograms).

Identification of 4-aminophenol in assays with cell extractsof Microbacterium sp. strain BR1. Additional experiments werecarried out in order to gain further insights into the degradation

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pathway of SMX. Resting-cell experiments with [14C]SMX, la-beled at its aniline moiety, showed the formation of additionaltransient and polar metabolites, albeit they did not accumulate toconcentrations allowing reliable mass spectrometric analysis (seeFig. S4 in the supplemental material). Therefore, we preparedcrude cell extracts of Microbacterium sp. strain BR1 and incubatedthem with 100 �M SMX along with the cofactors NADH andNADPH and without cofactors. 3A5MI was detected only whenNADH was used as the cofactor (45.5 �M [standard deviation{SD} � 3.6 �M]; n � 3). Concomitant with the formation of3A5MI, we observed the formation of an additional metabolite insuch incubation mixtures. The new metabolite was identified as4-aminophenol by comparison of its chromatographic behaviorand its mass spectrum (LC-MS) with those of an authentic stan-dard (HPLC mass spectra in Fig. 3a and b and LC-MS extractedion chromatograms in Fig. S5a and S5c in the supplemental ma-terial). Incubations of resting cells of Microbacterium sp. strainBR1 with 75 �M 4-aminophenol as a substrate led to a distinctdegradation of this compound compared to controls (see Fig. S6

in the supplemental material). Additionally, we confirmed4-aminophenol degradation by Microbacterium sp. strain BR1 bymeans of oxygen consumption rate measurements. In such exper-iments, we demonstrated that SMX-acclimatized cells had a sig-nificantly higher oxygen consumption rate in the presence of4-aminophenol than did nonamended controls (see Table S1 inthe supplemental material).

Degradation of SMX by cell extracts under an 18O2 atmo-sphere. In order to determine the type of enzyme activity involvedin the initial attack of SMX and the origin of the hydroxyl group of4-aminophenol, we incubated cell extracts of Microbacterium sp.strain BR1 with SMX and NADH under an 18O2 atmosphere.These assays led to the formation of 4-aminophenol giving a mo-lecular ion with a mass-to-charge ratio of 112 when analyzed byLC-MS in the positive-ionization mode. This ratio corresponds toa mass shift of the molecular ion by 2 atomic mass units in com-parison to that of controls incubated under a 16O2 atmosphere(HPLC mass spectra in Fig. 3b and c and LC-MS extracted ionchromatograms in Fig. S5c, S5d, S5e, and S5f in the supplemental

FIG 2 Degradation of different sulfonamides by resting cells of Microbacterium sp. strain BR1 and concomitant formation of metabolites (set 1). Shown are theconcentrations of the parent compound in the culture supernatant (circles) and the abiotic control (squares) and the concentrations of the correspondingmetabolite in the culture supernatant (triangles) and the abiotic control (inverted triangles). (a) Sulfadiazine and 2-aminopyrimidine; (b) sulfamethizole and5-methyl-1,3,4-thiadiazol-2-amine; (c) sulfamethoxazole and 3-amino-5-methylisoxazole; (d) sulfadimethoxine and 2,6-dimethoxy-4-pyrimidinamine; (e)sulfamethazine and 2,6-dimethyl-4-pyrimidinamine.

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material) and confirmed that the hydroxyl group introduced intothe aniline moiety originated from molecular oxygen.

Degradation of [14C]SDZ by resting cells of Microbacteriumsp. strain BR1. In order to obtain further evidence with regard to

the fate of the heterocyclic side chain and the sulfonyl moiety ofsulfonamides, a resting-cell experiment was carried out with[14C]SDZ (Fig. 1c), a different sulfonamide compound. SDZ was14C labeled at its heterocyclic moiety, allowing radioactive tracing

FIG 3 HPLC mass spectra of 4-aminophenol formed in supernatants after incubations of Microbacterium sp. strain BR1 cell extracts with NADH and SMX. (a)Authentic standard; (b) incubation under a 16O2 atmosphere; (c) incubation under an 18O2 atmosphere.

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of its fate during degradation. During incubation, we observedonly two 14C peaks upon analysis by means of HPLC coupled to aliquid scintillation radiodetector (LSRD) and HPLC-DAD (seeFig. S7 in the supplemental material). We identified these peaks as2-aminopyrimidine and the parent compound SDZ (Fig. 1d and c,respectively) by means of HPLC-DAD. These results showed thatSDZ was also degraded by Microbacterium sp. strain BR1 and thathere, analogously to the formation of 3A5MI in incubation mix-tures with SMX, 2-aminopyrimidine was formed as the sole me-tabolite that contained the 14C label of the original side group.Therefore, the data rule out the transient formation of side-chainmetabolites containing the sulfonyl moiety.

Sulfite formation during SMX degradation. In a further ex-periment with cell extracts of Microbacterium sp. strain BR1, wewere able to show that sulfite but not sulfate was formed concom-itantly with the degradation of SMX and the formation of 3A5MI.The concentrations of SMX and its metabolite 3A5MI, as well asthose of sulfite and sulfate, were monitored. SMX was degraded bythe cell extract at a rate of 1.85 �M min�1 (SD � 0.22 �M min�1),whereas 3A5MI and sulfite were formed at rates of 2.01 �M min�1

(SD � 0.05 �M min�1) and 1.92 �M min�1 (SD � 0.05 �Mmin�1), respectively (Fig. 4). In such incubation mixtures, we didnot observe the biotic formation of sulfate.

Degradation of different sulfonamides by SMX-adaptedresting cells of Microbacterium sp. strain BR1. In two incubationexperiments, we tested whether additional sulfonamides could bedegraded by Microbacterium sp. strain BR1 cells grown on SMX.In a first set of experiments, SMX, SDZ, sulfadimethoxine, sulfa-methazine, or sulfamethizole was added at a final concentration of100 �M to resting-cell suspensions with an OD600 of 0.5. All com-pounds were degraded, and metabolites corresponding to theaminated heteroatomic side group (3A5MI for SMX, 2-aminopy-rimidine for SDZ, 2,6-dimethoxypyrimidin-4-amine for sulfadi-methoxine [Fig. 1f and e, respectively], 2,6-dimethyl-4-pyrimidi-namine for sulfamethazine [Fig. 1h and g, respectively], and5-methyl-1,3,4-thiadiazol-2-amine for sulfamethizole [Fig. 1j andi, respectively]) accumulated in the assay mixtures (Fig. 2). Theycould be identified by comparing their respective retention timesand absorption spectra obtained by HPLC-DAD to those of theauthentic standards. The degradation rates of the sulfonamidescorresponded well to the formation rates of the respective metab-olites, with almost stoichiometric turnover. The highest degrada-tion rate was calculated for SDZ, at 2.51 �M min�1 g�1 (dryweight) (SD � 0.02 �M min�1 g�1 [dry weight]; n � 3), followedby sulfamethizole, at 2.26 �M min�1 g�1 (dry weight) (SD � 0.03

�M min�1 g�1 [dry weight]; n � 3). SMX was degraded at a rate of2.09 �M min�1 g�1 (dry weight) (SD � 0.02 �M min�1 g�1 [dryweight]; n � 3), and sulfadimethoxine and sulfamethazine weredegraded at lower rates of 1.64 �M min�1 g�1 (dry weight) (SD �0.07 �M min�1 g�1 [dry weight]; n � 3) and 1.53 �M min�1 g�1

(dry weight) (SD � 0.01 �M min�1 g�1 [dry weight]; n � 3),respectively.

In another experimental set, degradation of 100 �M asulamand 4-amino-N-phenylbenzenesulfonamide was tested by usingresting cells with an OD600 of 0.5 and compared to degradation ofSMX (Fig. 5). At a rate of 1.42 �M min�1 g�1 (dry weight) (SD �0.03 �M min�1 g�1 [dry weight]; n � 3), SMX was degradedslower than in the first set, while asulam was degraded at a rate of2.38 �M min�1 g�1 (dry weight) (SD � 0.07 �M min�1 g�1 [dryweight]; n � 3), and 4-amino-N-phenylbenzenesulfonamide wasdegraded at a rate of 0.90 �M min�1 g�1 (dry weight) (SD � 0.02�M min�1 g�1 [dry weight]; n � 3). Although lower degradationrates than those in the previous experiments were observed in thiscase, all compounds were completely degraded. As expected,4-amino-N-phenylbenzenesulfonamide led to the formation ofaniline, which was identified by comparing its retention time andabsorption spectrum obtained by HPLC-DAD to those of the au-thentic standard, which eventually was also degraded. Asulam wasdegraded when incubated with resting cells of strain BR1, but theexpected metabolite methylcarbamate was never detected. Never-theless, additional incubation experiments with asulam and acrude cell extract of Microbacterium sp. strain BR1 clearly showedthat sulfite was released concomitant to the degradation of theparent sulfonamide compound (see Fig. S8 in the supplementalmaterial). In a third experiment, resting cells with an OD600 of 7were incubated with 100 �M SMX, sulfanilamide, and 4-amino-N-cyclohexylbenzenesulfonamide, respectively. The higher celldensity was chosen, as preliminary tests did not lead to the degra-dation of the latter two compounds. While SMX was degraded asbefore (73.7 �M; SD � 0.9 �M), no degradation was observed forsulfanilamide and 4-amino-N-cyclohexylbenzenesulfonamide.

DISCUSSION

In this study, we show that Microbacterium sp. strain BR1 employsa novel pathway based on a type I ipso-substitution mechanism tometabolize sulfonamide antibiotics.

We demonstrated that upon incubation with SMX, Microbac-terium sp. strain BR1 produced 4-aminophenol as a transient me-tabolite, which was subsequently further degraded and concomi-tantly released sulfite and a dead-end metabolite, 3A5MI, inequimolar amounts. From these data, we conclude that SMX wasinitially hydroxylated at the ipso-position, forming 1-hydroxy-4-imino-N-(5-methylisoxazol-3-yl)cyclohexa-2,5-diene-1-sulfon-amide as an intermediate (compound b) (Fig. 6), which thenunderwent fragmentation to 4-iminocyclohexa-2,5-dienone (4-iminoquinone) (compound c) (Fig. 6), sulfur dioxide (compoundd) (Fig. 6), and 3A5MI (compound h) (Fig. 6).

Besides SMX, various other sulfonamides were metabolized byresting cells of Microbacterium sp. strain BR1 pregrown on SMX(Fig. 2 and 5). Noteworthy, only those sulfonamides were metab-olized for which the aminated side-chain fragments could delo-calize the pair of electrons coming from the heterocyclic cleavageof the amide bond and, therefore, were able to act as moderateleaving groups (shown for 3A5MI in Fig. 6). Sulfanilamide and4-amino-N-cyclohexylbenzenesulfonamide were not metabo-

FIG 4 Formation of 3-amino-5-methylisoxazole and sulfite during the deg-radation of SMX by cell extracts of Microbacterium sp. strain BR1. Shown arethe concentrations of SMX (circles), sulfite (triangles), net sulfate (invertedtriangles), and 3-amino-5-methylisoxazole (squares) over time.

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lized, in accordance with the notion that NH2� and cyclohexyl-

NH� are very poor leaving groups.At the moment, it remains unclear whether the substituent

spontaneously undergoes fragmentation once hydroxylation oc-curs or whether the reaction is enzymatically catalyzed. In anycase, this mechanism seems to be an effective strategy for micro-organisms to metabolize sulfonamides to innocuous compounds.So far, only biologically mediated ipso-hydroxylations have beendescribed for which the subsequently eliminated leaving groupsmaintained their molecular skeleton, and to that extent, the strat-egy elucidated here is novel to the best of our knowledge.

The hydroxyl group of 4-aminophenol originating from mo-lecular oxygen and the NADH-dependent enzyme activity arestrong indications that the hydroxylating enzyme activity was dueto a monooxygenase. Flavoprotein monooxygenases (33, 34) andcytochromes P450 (25) are known to catalyze electrophilic hy-droxylations of substituted aromatic compounds, whereby hy-droperoxo-flavin (35) and hydroperoxo-iron (44) species, respec-tively, act as the donor of electrophilic oxygen. It remains to beelucidated whether the hydroxylation activity of the Microbacte-rium sp. strain BR1 enzyme can be categorized as either one.

Sulfonamide antibiotics are used worldwide in large amounts(5, 6) and can be detected in the �g liter�1 range in various envi-ronmental matrices (36–38). Such concentrations, well below theinhibitory concentration, might still drive selection for utilizationas a carbon and energy source. Previous reports described theappearance of the aminated side chains as stable metabolites aftersulfonamide degradation, either with activated sludge (13) or, asrecently shown, with isolated bacteria (14, 15). A Microbacteriumsp., which has 99% identity to Microbacterium sp. strain BR1

based on 16S rRNA genes, was able to degrade sulfamethazineunder the concomitant release of 2,6-dimethyl-4-pyrimidinamine(14). Furthermore, Microbacterium lacus strain SDZm4 (99%identity to Microbacterium sp. strain BR1) was able to degradeSDZ under the concomitant release of 2-aminopyrimidine (15).These findings, together with the results presented here, lead tothe assumption that the isolates have a common degradationmechanism for sulfonamides, which may be transferable to othersludge or soil communities able to degrade sulfonamides.

This is the first publication examining this common degrada-tion mechanism and testing different sulfonamides with one iso-lated strain. The novel pathway described here is based on ipso-hydroxylation and subsequent fragmentation for the metabolismof sulfonamides. A better understanding of such reactions willhelp in appraising the risks underlying the increased appearanceof drug resistance in microorganisms (39). For instance, Mycobac-terium tuberculosis shows rather limited growth inhibition bySMX because it is able to disrupt the antibiotic activity of SMXthrough N-acetylation (40). Along these lines, it can safely be as-sumed that microorganisms able to metabolize sulfonamides bythe mechanism proposed here will also show decreased suscepti-bility to sulfonamide antibiotics. On the one hand, evolution andpropagation of novel microbial degradation pathways for pollut-ants will contribute to the elimination of such compounds fromthe environment and to the development of successful bioreme-diation strategies. To this extent, our findings can have an impacton the design of sulfonamide antibiotics, which are less persistentin the environment. On the other hand, if the target pollutantshappen to be antimicrobials, there will also be a certain risk thatsuch novel metabolic traits may contribute to drug resistance. As

FIG 5 Degradation of different sulfonamides by resting cells of Microbacterium sp. strain BR1 and concomitant formation of metabolites (set 2). Shown are theconcentrations of the parent compound in the culture supernatant (circles) and the abiotic control (squares) and the concentrations of the correspondingmetabolite in the culture supernatant (triangles) and the abiotic control (inverted triangles). (a) SMX and 3-amino-5-methylisoxazole; (b) 4-amino-N-phenylbenzenesulfonamide and aniline; (c) asulam.

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of now, sulfonamide resistance has often been attributed to thepresence of sul genes, coding for an alternative dihydropteroatesynthase to alleviate inhibition of the folic acid biosynthesis path-way targeted by sulfonamides (41–43). However, the catabolismof sulfonamides may present a parallel resistance mechanism thathas so far been overlooked. Further research will have to addressmore intensely the complex relationship between microbial me-tabolism of antimicrobial compounds at low concentrations andthe development and propagation of resistance mechanisms de-rived from such metabolic traits.

ACKNOWLEDGMENTS

This work was supported by the European Commission, which funded theproject MINOTAURUS under grant agreement number 265946 in the 7thFramework Programme, and Swiss National Science Foundation grantnumber 310030_146927.

We thank Burkhard Schmidt (RWTH Aachen University) for kindlyproviding a sample of 14C-labeled sulfadiazine, Nora Corvini Boussouelfor assistance in culture preparation, Erik Ammann and Gregor Hommesfor assistance with the Seahorse system, and Andy Brown for Englishcorrections.

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FIG 6 Proposed mechanism for the degradation of SMX by Microbacterium sp. strain BR1. SMX is hydroxylated. Note that [FeIII-O-OH] represents thehydroperoxo-iron of a P450-dependent monooxygenase, which has been chosen arbitrarily as an electron donor (compound a), followed by electron rearrange-ment (compound b). This results in a concerted cleavage, giving rise to the formation of benzoquinone-imine (compound c), sulfur dioxide (compound d), anda 3-imino-5-methylisoxazole intermediate (compound e). Reduction of compound c yields 4-aminophenol (compound f), while compound d is hydrated toform sulfite (compound g), and compound e is transformed to 3-amino-5-methylisoxazole by accepting a proton.

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