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Applied and Environmental Microbiology, April 1999, p. 1556-1563, Vol. 65, No. 4
Animal Health
Research,1 Pharma
Research,2 and Central
Research,
Received 1 July 1998/Accepted 28 January 1999
Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is
widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring 14CO2 production from [14C]CIP in
liquid cultures. Sixteen species inhabiting wood, soil, humus, or
animal dung produced up to 35% 14CO2 during 8 weeks of incubation. Despite some low rates of
14CO2 formation, all species tested had reduced
the antibacterial activity of CIP in supernatants to between 0 and 33%
after 13 weeks. Gloeophyllum striatum was used to identify
the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10% 14CO2 from C-4 and the piperazinyl
moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The
structures of 11 metabolites were elucidated by
high-performance liquid chromatography combined with electrospray
ionization mass spectrometry and 1H nuclear magnetic
resonance spectroscopy. They fell into four categories as follows: (i)
monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an
isatin-type compound, proving elimination of C-2, and (iv)
metabolites indicating both elimination and degradation of the
piperazinyl moiety. A metabolic scheme previously described for
enrofloxacin degradation could be confirmed and extended. A new type of
metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory
evidence for the proposed network of congeners. This may result from
sequential hydroxylation of CIP and its congeners by hydroxyl radicals.
Our findings reveal for the first time the widespread potential for CIP
degradation among basidiomycetes inhabiting various environments,
including agricultural soils and animal dung.
Being active against many
gram-negative and gram-positive pathogenic bacterial species,
fluoroquinolones (FQs) have found wide application in human
medicine. Ciprofloxacin (CIP [Fig. 1]) is used to treat infections of the urinary, respiratory, and
gastrointestinal tracts (23). During its passage
through the human body, CIP can be metabolized by sulfation and, to
a limited extent, by oxidation of its piperazine moiety
(4). Similar metabolites as well as glucuronidation, but not
degradation of the heterocyclic core, have been observed in various
animal species (4). A significant quantity of an FQ
may be excreted unchanged and introduced into the environment
through wastewater, predominantly from clinical settings
(11). However, CIP and other FQs were shown to be tightly bound by human feces (6, 26) and soil (18, 22,
27), being no longer bioavailable and, hence, are unlikely to
exert a significant selection pressure (14, 20, 26, 27). On the other hand, strong binding may delay biodegradation and could partly explain the supposed recalcitrance of FQs (12, 18, 21). Their fate in sewage sludge which, if not burned or
deposited in landfills, may be further processed by composting, is
unknown.
Only a few fluorinated aliphatic compounds such as poisonous
fluoroacetate occur in some species of plants and streptomycetes. Fluorinated aromatics, as exemplified by FQs, are not found among natural products (10). This raised concern about their
biodegradability and environmental impact (21). Recently, we
have shown in vitro degradation of the veterinary FQ
enrofloxacin (EFL) by basidiomycetes, notably, the brown rot fungus
Gloeophyllum striatum as well as three species of white rot
fungi (19). G. striatum was most active, evolving
up to 50% 14CO2 within 8 weeks from
[4-14C]EFL bound to wheat straw. EFL, preadsorbed to
agricultural soil, could also be mineralized, although at a much lower
rate (19). Metabolites formed from [4-14C]EFL
by G. striatum in liquid cultures included various mono- and
dihydroxylated congeners as well as compounds indicating the cleavage
of both its heterocyclic core and amine moiety (31).
White rot fungi are able to degrade the lignin of woody plant cell
walls and, in addition, a broad spectrum of pollutants (2).
These activities are attributed to extracellular ligninolytic enzymes
such as lignin peroxidase, manganese-dependent peroxidase, and laccase.
Such enzymes catalyze degradation via diffusible oxidizing agents,
e.g., aryloxy radicals, Mn3+, or specific mediators
(4a, 7, 9, 13). In contrast, brown rot fungi appear to
preferentially degrade the cellulose and hemicellulose components of
wood, while lignin is modified by hydroxylation and demethylation and,
to a minor extent, by depolymerization (9). As G. striatum did not exhibit peroxidase or laccase activity, a
hydroxyl radical-based degradation mechanism was postulated to be
operative (31). The involvement of a Fenton-type reaction in
wood degradation was first proposed by Koenigs (17). Hydroxyl radicals were thought to be generated through reduction of
hydrogen peroxide by ferrous iron (1, 8, 9, 15, 17) as
follows: Fe2+ + H2O2 The aim of our study was (i) to assess inactivation and
degradation of CIP by G. striatum, (ii) to identify
metabolites formed from CIP, verifying and possibly extending the
metabolic scheme described for EFL (31), and (iii) to test
basidiomycetes inhabiting ecologically relevant sites such as
agricultural soils and pastures for FQ degradation potential.
Preliminary results have already been reported ([29, 32]).
Organisms.
G. striatum DSM 9592, isolated by M. Capelari in May 1992, in Assis, São Paulo, Brazil, served as the
primary model organism, while G. striatum DSM 10335 was
employed as a reference (19). Cultures of the other species
listed in Table 1 were (i) obtained from
the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany, (ii) isolated from fruiting bodies collected by
M. Stadler (strain designation, WP), or (iii) provided by W. Fritsche,
Jena, Germany (Agrocybe praecox P1). A taxonomic evaluation of the latter strains will be reported elsewhere (24).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Degradation of Ciprofloxacin by Basidiomycetes and Identification
of Metabolites Generated by the Brown Rot Fungus
Gloeophyllum striatum
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (10K):
[in a new window]
FIG. 1.
Molecular structure of ciprofloxacin and positions of
the 14C label in [4-14C]CIP (*) and
[piperazine-2,3-14C]CIP (+).
Fe3+ + HO· + HO
.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Degradation of ciprofloxacin in various taxonomic groups
of basidiomycota
Culture conditions. Media and culture conditions enhancing FQ degradation were identical to those reported previously (31), except that the concentration of Mn2+ in mineral medium was increased from 2 to 20 µM (except for G. striatum). Briefly, the organisms were precultured unagitated in 30 ml of malt medium at room temperature for 7 days. Then, mycelia were washed and transferred into a defined mineral medium devoid of carbon, nitrogen, and phosphate at substrate concentrations.
Ciprofloxacin and reference compounds.
The chemical
structure of CIP
[1-cy-clopropyl-7-(1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid],
including the positions of 14C labeling, is shown in Fig.
1. The nonlabeled standard compound had a chemical purity of >99.9%.
Chemically synthesized references (Table
2) were kindly provided by W. Hallenbach
and U. Petersen (Bayer AG, Leverkusen, Germany).
[4-14C]CIP was synthesized by R. Koch (Bayer Corp.,
Stilwell, Kans.), and [piperazine-2,3-14C]CIP was
synthesized by M. Conrad (Bayer AG, Wuppertal, Germany). Both compounds
were purified immediately before use by R. Thomas (Bayer AG,
Wuppertal). The specific activities of
[4-14C]CIP-hydrochloride and
[piperazine-2,3-14C]CIP-hydrochloride were 6.13 and 6.96 MBq/mg, while their radiochemical purities were >97% and >99%,
respectively, as determined by high-performance liquid chromatography
(HPLC).
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Experimental procedures. Test system A, which was described in detail before (31), was employed during screening for degradation of CIP. G. striatum had a concentration of approximately 2 mg (dry weight) of mycelium per ml. For all other organisms, the mycelial mass obtained after 7 days of growth in malt broth was washed and transferred into mineral medium. Each flask received either [4-14C]CIP (7.3 kBq) or [piperazine-2,3-14C]CIP (9.3 kBq), which was supplemented with nonlabeled drug to give a final concentration of 10 ppm. All experiments were carried out at room temperature in the dark to prevent photodegradation of CIP.
Test system B was utilized to produce metabolites (31). Each 250-ml Erlenmeyer flask contained 30 ml of mineral medium, G. striatum DSM 9592 (as mentioned above), and 900 µg of CIP, 14C labeled with 400.6 or 890.3 kBq of [4-14C]CIP or [piperazine-2,3-14C]CIP, respectively. The vessels were kept at 150 rpm. Produced 14CO2 was trapped in NaOH and determined by liquid scintillation counting as described before (31). For structure elucidation, four supernatants of 90-h-old cultures containing [4-14C]CIP were combined and lyophilized. After resuspension in 2 ml of water, this preparation was fractionated by micropreparative HPLC.Analytical and micropreparative HPLC. HPLC was performed as described previously (31), although the gradients had to be modified. The eluent was composed of 0.1 mM ammonium formate in 1% formic acid (component A) and acetonitrile (component B). For analytical purposes, gradient method I was employed as follows. Starting at 100% A for 2 min, A was linearly decreased to 94% over 5 min and then to 88% over a further 10 min. Thereafter, A was kept constant for 15 min and then decreased to 72% over 5 min and, finally, to 0% over 10 min. Method II was used for micropreparative isolation of metabolites. Component A contained an additional 1% (vol/vol) 2-propanol. After 2 min, A was decreased to 97% over 3 min and then to 90% over 20 min. Following a decrease to 87% over 18 min, A was reduced to 68% over 10 min and to 0% over 7 min. In order to separate metabolite F-6 from CIP, method II was modified, giving method III, as follows. First, the column temperature was reduced from 30 to 9°C. After 2 min, A was successively decreased to 94% over 3 min, to 92% over 10 min, to 82% over 55 min, to 70% over 10 min, and to 0% over 10 min. The flow rate was 1 ml/min.
Purification and derivation of F-10. A solid-phase cartridge containing 400 mg of Adsorbex SPE-RP18 (particle size, 40 to 63 µm; Merck, Darmstadt, Germany) was conditioned by washing it twice with 5 ml of methanol followed by 5 ml of 1% aqueous formic acid. Five milliliters of supernatant from a culture of G. striatum degrading [piperazine-2,3-14C]CIP was passed through this device. The effluent volume (containing F-10) was reduced to about 100 µl by freeze drying. To this preparation, 20 µl of borate buffer pH 9.2 (Merck) was added, followed by 50 µl of 0.5% (wt/vol) 2,4-dinitro-1-fluorobenzene in acetonitrile. This solution was incubated at 95°C for 5 min.
Other analytical techniques. HPLC-electrospray ionization mass spectrometry (HPLC-MS), 1H nuclear magnetic resonance spectroscopy (1H-NMR), and liquid scintillation counting were performed as described in reference 31. High-resolution electrospray mass spectrometry (HR-ESI-MS) was performed on a MAT 900 mass spectrometer (Finnigan-MAT, Bremen, Germany) operated at a resolution of 6,000 (10% valley definition) applying the peak matching mode. The spray needle voltage was 3.8 kV. Nitrogen at 100 kPa served as sheath gas. The capillary was held at 250°C. Samples spiked with the calibration standard, polypropylene glycol 425 (Sigma-Aldrich Chemie, Deisenhofen, Germany), were delivered at a flow rate of 10 µl/min by an infusion pump (no. 22; Harvard Apparatus, Inc., South Natick, Mass.).
Residual antibacterial activity. Activity of CIP in supernatants of liquid cultures was determined by an agar diffusion procedure with Balanced Sensitivity Test Agar (Difco, Augsburg, Germany) and Escherichia coli ATCC 8739 (MIC, = 0.015 µg of CIP/ml) as the test organism. Samples of 0.1 ml, drawn from quadruplicate cultures of selected fungal strains (Table 1), were transferred into agar wells (diameter, 9.5 mm). Five replicate serial dilutions of a CIP standard were used to construct a reference curve correlating the concentration of CIP (0.05 to 10 µg/ml) with the respective zone of growth inhibition (11 to 34 mm) after 20 h at 37°C.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Kinetics of 14CO2 formation from [14C]CIP. Precultured mycelia of G. striatum DSM 9592 were resuspended in a defined mineral medium containing 10 ppm CIP 14C labeled either at C-4 or the piperazine moiety (Fig. 1). After 8 weeks, 17.0% ± 2.1% and 10.1% ± 1.0% 14CO2 were produced from both label positions, respectively (Fig. 2). The kinetics were similar to those determined for EFL (31). Cultures of G. striatum DSM 10335 evolved 10.6% ± 3.8% and 7.2% ± 0.6% 14CO2 from the respective compounds. Other species of basidiomycetes, inhabiting either wood, humus, agricultural soils, pastures, or even cattle dung, had formed between 0.7 and 24.3% 14CO2 from [4-14C]CIP after 8 weeks, while 2.2 to 35.3% 14CO2 had been produced from [piperazine-2,3-14C]CIP (Table 1). Despite the relatively small amounts of 14CO2 evolved by some species, all those tested had lowered the apparent residual antibacterial activity of CIP in supernatants to between 0 and 33% ± 21% after 13 weeks (Table 1). Most remarkably, CIP was completely inactivated by the soil-inhabiting fungus Clitocybe odora.
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, 
)
and DSM 10335 (
, 
). Mycelia were incubated in a defined mineral
medium containing 10 ppm CIP labeled either at C-4 (Metabolites in supernatants of G. striatum. Typical HPLC elution profiles obtained from cultures of G. striatum DSM 9592 degrading either [4-14C]CIP or [piperazine-2,3-14C]CIP are shown in Fig. 3. Identical profiles were found for G. striatum DSM 10335 (data not shown). A reference profile, already reported for EFL (31), was included (Fig. 3C) to facilitate a provisional assignment of the metabolites. Molecular weights of major metabolites were determined by HPLC-MS (Table 3) and are also included in Fig. 3. An overall similarity in the profiles is obvious, although the peaks of metabolites generated from CIP were shifted toward the more polar side of the gradient. However, metabolite F-8 had a similar retention time and an identical molecular weight, regardless of whether its source had been CIP or EFL. F-8 was absent from the HPLC trace when piperazine-labeled CIP was used as the substrate (Fig. 3, trace A). Again, a broad, tailing, and oversized peak (designated F-10) appeared at the front of trace A. F-10 was retained by adsorption to the solid-state scintillator of the detector cell, thereby causing such an artificially enlarged signal (31). Micropreparative separation of F-10 and subsequent liquid scintillation counting of the fractions indicated that F-10 accounted for approximately 23% of the initially applied radioactivity.
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Structure determination. Major metabolites were isolated from supernatants of cultures of G. striatum DSM 9592 in which [4-14C]CIP had been degraded. Their systematic names are given in Table 2; chemical structures are shown in Fig. 4. Retention times, pseudomolecular ions, and characteristics of their UV absorption spectra are compiled in Table 3. Metabolites were assigned to four categories as follows.
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(i) Monohydroxylated congeners.
Metabolites F-1 and F-2 (Table
2) were first characterized by their UV spectra and molecular weights,
303 and 329, respectively (Table 3). The 1H-NMR
spectrum of F-1 showed a set of signals which was almost identical to
those of CIP, except for an upfield shift of the signal of H-2 (Table
4). This indicated the replacement of the carboxyl group with a hydroxyl group (31). The
1H-NMR spectrum of F-2 (Table 4) revealed singlets of H-5
and H-8, proving the elimination of fluorine (Fig. 4). To isolate F-6
(Table 2) by HPLC, the elution gradient had to be run at lowered
temperature. Characteristic UV absorption maxima of F-6, at 242 and 331 nm (Table 3), were almost identical to those observed with F-6 derived
from EFL (31). The molecular weight of F-6, 347 (Table 3),
suggested monohydroxylation. Its 1H-NMR spectrum contained
only one doublet with an H,F coupling constant (J = 11.3 Hz), indicating a proton in the ortho position to
fluorine (Table 4). Therefore, hydroxylation had occurred at C-8 (Fig.
4).
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(ii) Dihydroxylated congeners. Metabolites F-5 and F-8 (Table 2) had molecular weights of 345 and 279, respectively (Table 3). The 1H-NMR spectrum of F-5 contained only two singlets, one of H-2 and another (7.29 ppm) which could have been derived from either H-5 or H-8. Both moieties, piperazinyl and cyclopropyl, were also detected (Table 4). Hence, two alternative structures were feasible, either a 5,6-dihydroxylated or a 6,8-dihydroxylated congener (Fig. 4), as observed for EFL (31). The 1H-NMR spectrum of F-8 was devoid of signals of the piperazinyl moiety. Furthermore, signals of H-2, H-5, and the cyclopropyl group showed chemical shifts and multiplicities identical to those of the reference compound (data not shown). Identification of F-7 (Table 2), due to its instability under our analytical conditions, had to be based on molecular weight (373, suggesting dihydroxylation) and UV absorption spectrum (Table 3). The latter was almost identical to the spectrum observed for F-7 of EFL (31), suggesting identical substitution patterns of both heterocyclic cores.
(iii) Isatin-type congener. F-3 was present in supernatants at a low concentration, as observed before with EFL (31). However, it could be identified by its ion m/z 290, which was accompanied by m/z 292 generated from [4-14C]CIP (Table 3), as well as by m/z 292 plus 294, if [piperazine-2,3-14C]CIP had served as the substrate. Furthermore, these ions were detected at a relative retention time (between F-2 and F-4) similar to that observed for F-3 derived from EFL and moxifloxacin (30, 31).
(iv) Metabolites indicating either elimination or oxidation of the piperazine moiety. Due to its polarity, F-10 (piperazine) was hardly retained by the HPLC column. However, piperazine derivatized to give 2,4-dinitro-1-(1-piperazinyl)-benzene was eluted at 20.8 min and could be identified by HPLC-MS. Hence, F-10 was purified from a culture containing degraded [piperazine-2,3-14C]CIP by removing all other major metabolites by solid-phase extraction. After derivation of F-10, the products comprised an ion at m/z 253, which was accompanied by a specific ion pattern (253/255/257 = 10:1:3) caused by the ratio of 12C and 14C atoms (either zero, one, or two) contained in the piperazine moiety. A corresponding pattern was found in [piperazine-2,3-14C]CIP at m/z 332, 334, and 336, proving that F-10 was a specific derivative thereof. Other polar metabolites were not detected. Metabolites F-4 and F-9 (Table 2) were identified by cochromatography by using synthetically prepared reference compounds as standards (Table 3).
A new metabolite, F-12 (Table 2), reached only a low concentration in supernatants. However, after purification, a 1H-NMR spectrum was obtained (Table 4). It contained three singlets (H-2, H-5, and H-8), the latter ones indicating elimination of fluorine. Furthermore, the integral for methylene groups assigned to the piperazinyl moiety was reduced from 8 to 4 (Table 4), in accordance with the elimination of one ethylidene bridge. This was in agreement with its molecular formula, C15H17N3O4, determined by HR-ESI-MS; the measured mass of [M + H]+, m/z 304.1296, closely matched the theoretical value of 304.1297. The resulting structure is shown in Fig. 4.Metabolites at trace concentrations. Due to their extremely low concentrations, five additional metabolites of CIP could be detected by HPLC-MS only. All had retained both types of 14C labels and showed specific patterns of pseudomolecular ions. Their hypothetical structures are included in Fig. 4. Oxidative decarboxylation of F-2, F-4, and F-5 would give rise to molecular weights of 301, 277, and 317, respectively. Hydroxylation of F-1 and F-4 at either position C-5 or C-8 would generate molecular masses of 319 and 321 Da, respectively.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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G. striatum and other species of basidiomycetes indigenous to agriculturally relevant sites were shown to degrade CIP in liquid cultures. The actual extent of inactivation, with G. striatum, for example, could be related to either (i) the amount of 14CO2 produced, e.g., 17% from [4-14C]CIP within 8 weeks, or (ii) the residual concentration of CIP, e.g., at the time of harvest, 90 h. At that point, identified metabolites represented at least 50% of the applied 14C label. Thirdly, for selected species, residual antibacterial activity in supernatants was directly measured by employing an agar diffusion assay (see below).
Rate and extent of 14CO2 production from [4-14C]CIP by G. striatum were apparently twice as high as those from [piperazine-2,3-14C]CIP. The piperazine moiety contains two equivalent ethylidene bridges which are most likely targeted with similar probability. Because the 14C label is located exclusively at positions C-2' and C-3' (Fig. 1), produced 14CO2 represents only half of the total ethylidene groups degraded. This explains why metabolite F-4 is detectable at approximately half the relative peak area (compared, e.g., with F-2) in HPLC profiles obtained from supernatants containing degraded [piperazine-2,3-14C]CIP (Fig. 3, traces A and B; see also in reference 31, Fig. 4, traces A and C). Therefore, the rates of 14CO2 formation from C-4 and piperazine-labeled CIP actually indicate that both parts of the molecule, the heteroaromatic core and the aliphatic substituent, were degraded by G. striatum at approximately the same rate.
Identification of metabolites and proposed metabolic scheme. HPLC profiles of metabolites present in 90-h-old supernatants of cultures of G. striatum (Fig. 3) resembled profiles observed for EFL (31), although metabolite peaks were shifted toward lower retention times. One metabolite was common to CIP and EFL, F-8 (Fig. 4), which had lost its amine moiety (Fig. 3, traces B and C). The turnover of CIP could not be followed directly because, under analytical HPLC conditions, a major metabolite, F-6, was not separable. However, after about 3 weeks, CIP was almost quantitatively transformed into congeners. After purification, metabolites were characterized by combining cochromatography, UV spectroscopy, HPLC-MS, and 1H-NMR spectroscopy. The molecular structures of 6 of 11 metabolites (F-1, F-2, F-4, F-5, F-6, and F-12) were proved by complete 1H-NMR spectra.
The metabolic scheme depicted in Fig. 4 shows four principal degradation routes (A through D) which, due to similar concentrations of primary metabolites (F-1, F-2, F-6, and F-4), appear to be simultaneously employed. They may reflect different sites of initial attack of CIP by hydroxyl radicals. Either reaction, i.e., oxidative decarboxylation, defluorination, hydroxylation at C-8, or oxidation of the amine moiety, will terminate antibacterial activity of CIP (4, 5, 31, 33). Because each primary metabolite offers several sites for further attack, an extensive branching of the basic routes can be expected, resulting in a network of metabolites. Dihydroxylated metabolites, contained in routes A, B, and C, were quite unstable. Tentatively identified trace metabolites (included in Fig. 4) could have been generated either by one-step reactions from firmly identified congeners of CIP or by alternative reaction sequences. An isatin-type derivative, F-3, proved the elimination of C-2 and suggested an intermittent cleavage of the heterocyclic core of CIP. Isatin is a known intermediate in fungal degradation pathways for indole and tryptophan. Its multiple effects on fungal metabolism have been reviewed previously (16). Hydroxylation of F-6 at C-7 caused elimination of the piperazine moiety, F-10. Such elimination is also feasible for several other metabolites, which may explain the concentration of 23% detected for F-10 at 90 h. Therefore, F-10 was an important indicator of CIP inactivation, compared with approximately 3% 14CO2, which had been formed from [4-14C]CIP at that time. Metabolites homologous to F-4 and F-9 are common in mammals (4). Recently, a variety of soil microbes was also demonstrated to produce F-9 from danofloxacin (3). Such metabolites were reported to have a residual antibacterial activity on the order of
3% compared
with CIP (4, 33). Hence, they are also important indicators
of inactivation of FQs. Most notably, metabolite F-12 was identified
here for the first time. It may be formed from F-4 by hydroxylation or
from F-2 in a multistep oxidation process. Therefore, it links the
principal degradation routes B and D. Overall, the proposed metabolic
scheme closely resembles those proposed for EFL and moxifloxacin
(30, 31). Our results may further strengthen the hypothesis
that brown rot fungi such as G. striatum are able
to perform Fenton's reaction in order to produce hydroxyl radicals
(8, 9, 15, 17). Other mechanisms employed by bacteria and
fungi in aerobic degradation of N-heterocyclic compounds
have been discussed before (31).
Approaching field conditions from in vitro
experiments.
Drug residues, excreted by patients under FQ
therapy, enter the environment via wastewater (11) and,
theoretically, by processed sewage sludge. Unspecific binding to feces
(6, 26) and soils (18, 22) restricts the mobility
of FQs in the environment and greatly reduces their bioavailability
(20, 27). At present, it is unknown whether FQs are degraded
during anaerobic and, more specifically, aerobic process steps in
sewage sludge treatments. However, FQ degradation is likely to occur
during composting procedures. Our screening experiments demonstrated
that coprophilous basidiomycetes, such as Cyathus
stercoreus, as well as six species inhabiting various soil
compartments, have the potential to degrade CIP. All species evolved
14CO2 from CIP labeled at either C-4 or the
piperazine moiety. Standard deviations indicated considerable variation
in the performance of individual cultures. Moreover, the variable
activities between species most likely reflected inappropriate culture
conditions rather than principally different degradation potentials.
Certainly, the specificity of some relatively low activities needs to
be confirmed, after improved culture conditions have become available; such work is currently in progress (28). Despite these
reservations, a high inactivation potential for CIP was present in all
basidiomycetes tested. Notably, no residual antibacterial activity
remained in the supernatant of C. odora after 13 weeks
(Table 1). In more recent studies, a residual activity of 1% has
been determined in 4-week-old cultures of G. striatum DSM 9592 decomposing EFL (28).
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ACKNOWLEDGMENTS |
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We thank our colleagues at various chemistry departments at Bayer for providing the labeled and nonlabeled standard compounds and S. Ochtrop, A. Gerhardt, and J. Schneider for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Bayer AG, Building 6700, D-51368 Leverkusen, Germany. Phone: 49 2173 38 4882. Fax: 49 2173 38 3766. E-mail: heinz-georg.wetzstein.hw{at}bayer-ag.de.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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