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Applied and Environmental Microbiology, October 2008, p. 6147-6150, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00516-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transformation of N-Phenylpiperazine by Mixed Cultures from a Municipal Wastewater Treatment Plant

Carina M. Jung,¹ Thomas M. Heinze,² Joanna Deck,¹ Ruth Strakosha,^1, and John B. Sutherland^1*

Divisions of Microbiology,¹ Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas²

Received 3 March 2008/ Accepted 28 July 2008

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Samples from a wastewater treatment plant were used as inocula for mixed cultures dosed with N-phenylpiperazine (NPP), a model compound containing the piperazine ring found in many fluoroquinolones. Chemical analyses showed that NPP (50 mg liter^–1) disappeared in 12 days, with the appearance of a transient metabolite and two nitrosated compounds.

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Municipal wastewater treatment plants (WWTPs) represent reservoirs for antimicrobial-resistant organisms and resistance genes that may spread by lateral gene transfer (4, 24). Fluoroquinolone antimicrobials have been detected in WWTPs (6, 11, 14, 17, 22), where most are expected to bind to sludge (6), but some may be released via the final effluent (6, 11, 22) into the aquatic environment (8, 13, 26). One strategy to limit proliferation of fluoroquinolone-resistant bacteria is to enhance biodegradation. Although fluoroquinolones are transformed by some fungi (10, 15, 19, 21, 27, 28), information on bacterial transformation is limited (5). Some bacteria modify ciprofloxacin and norfloxacin by N-acetylation or N-nitrosation, with no carbon loss (2, 3, 12, 16, 18, 23) but reduction of antibacterial activity (2). The initial site of transformation of these fluoroquinolones is nearly always the piperazine moiety. Our intention was to test the ability of WWTP microorganisms to metabolize the piperazine ring in a model compound for fluoroquinolones.

Grab samples of aerobic reactor liquor and final effluent were taken from the Little Rock Wastewater Utility Treatment Plant (Little Rock, AR) in August 2006. Solid-phase extraction was performed on subsamples (2, 9), using 3-ml Waters Sep-Pak C₁₈ cartridges with glass wool as a prefilter. The eluate was dried in vacuo, resuspended in 1 ml methanol, and analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) (2) with parent masses alternated between protonated molecules of fluoroquinolones and Q3 scanned from m/z 200 to 400 in 0.2 s (9). Ciprofloxacin was detected in the liquor at 750 ng liter^–1. Either ofloxacin or levofloxacin (not distinguishable under our conditions) was detected in the liquor at 500 ng liter^–1 and in the effluent at 24 ng liter^–1; the levels were consistent with other studies (6, 7, 11, 14).

Assuming that a site containing fluoroquinolones may also contain organisms capable of degrading these and similar compounds, we established mixed cultures from the liquor of the WWTP aerobic reactor. A minimal salts medium containing (per liter) 1 g KH₂PO₄, 1 g (NH₄)₂HPO₄, 0.88 g NaNO₃, 0.20 g MgSO₄·7H₂O, and 0.10 g B-D Difco yeast nitrogen base was brought to pH 7 with KOH and autoclaved. One-milliliter volumes of sterile 0.15 M FeCl₃ and 0.18 M CaCl₂ were added after cooling. Degradation of a model compound, N-phenylpiperazine (NPP) (1), by the mixed cultures was analyzed in lieu of fluoroquinolones, since it contains the piperazine ring found in many fluoroquinolones that is most often the site of initial enzymatic attack. Triplicate 50-ml volumes were inoculated with a final concentration of 0.5% (vol/vol) liquor. Cultures and triplicate sterile controls were dosed with filter-sterilized NPP (100 mg liter^–1; Sigma); triplicate control cultures did not receive NPP. All flasks were incubated at 30°C with shaking at 200 rpm. Two ml was removed from each flask at each sampling time and adjusted above pH 8.5 with KOH. Samples were extracted three times with 2 ml ethyl acetate. The extracts were combined, dried in vacuo, and reconstituted with 400 µl methanol for analysis.

Twenty µl of prepared sample was injected into an Agilent Technologies 1100 high-performance liquid chromatography (HPLC) apparatus using an Inertsil 5-µm ODS-3 column (250 by 4.6 mm; Phenomenex). The mobile phase consisted of a gradient of H₂O-methanol (each with 10 mM NaOH) that increased from 25 to 75% methanol over 15 min, then from 75 to 100% over the next 5 min, and then was held at 100% for 5 min. The flow rate was 1 ml min^–1 for the first 20 min and then 1.5 ml min^–1 for 5 min. The diode array signal was set at 254 nm with a reference of 360 nm. External standard curves were generated for each substrate.

For LC/ESI-MS, full scans were acquired from m/z 90 to 550 in 1 s. For MS/MS identification of metabolites, the electrospray capillary temperature was 300°C and other conditions were the same as above. The mobile phase was a linear gradient at 0.2 ml min^–1 of either 5 to 95% acetonitrile in water over 40 min with constant 21.7 mM formic acid (pH 3) or 5 to 90% of the same with constant 10 mM ammonium acetate (pH 7). Comparisons of retention times, UV spectra, and mass and product-ion spectra with those of standards resulted in identification, which was confirmed by ¹H nuclear magnetic resonance (NMR) spectroscopy (1, 20).

When NPP (100 mg liter^–1) was introduced as the sole carbon source, HPLC showed a reduction in the NPP peak and the appearance of new peaks at about 21 days. This trend continued through 42 days in the mixed cultures (Fig. 1A), but NPP remained essentially unchanged in sterile controls (Fig. 1B). The loss of NPP in 42 days did not exceed 50%. Mass spectrometric analysis of successfully degrading cultures at 21 days revealed a transient metabolite, peak I (Table 1), which was identified by NMR (Table 2), and an authentic standard (Aldrich) as N-phenylethylenediamine (PED) (Fig. 2). Peak I had largely disappeared by 42 days (Fig. 1A).

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FIG. 1. Representative total ion chromatograms, obtained by LC-MS for N-phenylpiperazine and the metabolites produced from it in 42 days. (A) Mixed WWTP reactor liquor cultures first produced the compound represented by peak I, which was found at higher concentrations at earlier sampling times, and then the compounds represented by peaks II and IV. (B) Sterile controls accumulated small amounts of two compounds eluting together in peak III. The percentage of each peak compared to that of NPP in sterile controls is shown in parentheses.

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TABLE 1. Retention times and mass spectra (LC/ESI-MS/MS) for metabolites from N-phenylpiperazine-transforming mixed cultures

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TABLE 2. 1H-NMR data (500.13 MHz) for metabolites produced by mixed cultures in minimal salts medium containing N-phenylpiperazine

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FIG. 2. Structures of ciprofloxacin, ofloxacin, N-phenylpiperazine, and the three NPP transformation products identified in mixed WWTP liquor cultures.

Two major transformation products (peaks II and IV in Fig. 1) were identified as N-nitroso-PED and N-nitroso-NPP, respectively (Tables 1 and 2; Fig. 2). Although the analyte of peak IV was relatively stable, that of peak II was unstable and required further confirmation. N-Nitroso-PED was synthesized by dissolving 10 mg PED and an equimolar amount of NaNO₂ in 2 ml H₂O and purging with argon for 2 min. HCl was added until a pH of 5 was reached, and then the solution was stirred for 2 h (25).

Authentic PED was then introduced at 100 mg liter^–1 into mixed cultures as a sole carbon source. Analysis by HPLC at 14 days showed no change in the PED substrate, but at 34 days it had completely disappeared, with the appearance of a single metabolite corresponding to peak II (Fig. 1A). Direct exposure probe MS suggested a molecular weight of 165; the electrospray mass spectrum had ions at m/z 147, 136, and 106. The expected protonated molecule (m/z 166) was not observed, most likely due to instability. Product-ion spectra of the ions at m/z 147 and 136 were identical, with a major ion at m/z 106 and minor ions at m/z 79 and 77. Data for peak II (Table 1) were compared to those for synthetic N-nitroso-PED. They showed identical retention times, ESI mass spectra, product-ion mass spectra, and UV spectra at both pH 3 and pH 7. The chemical shifts of the NMR results for peak II (Table 2) were the same as those for the synthetic compound, further supporting the identification of N-nitroso-PED.

N-Acetyl-NPP and N-formyl-NPP (both eluting in peak III) (Fig. 1) were detected in cultures and sterile controls at levels of usually less than 1% of the starting substrate; the source is unknown.

In a separate experiment, molasses (Grandma's unsulfured) was added as a supplementary carbon source at 0.4% (vol/vol). WWTP liquor was inoculated for a final A₆₀₀ of 0.02. Cultures were dosed with either NPP (50 or 100 mg liter^–1) or PED (100 mg liter^–1). To avoid losing organic-insoluble metabolites, 0.1-ml samples were taken at intervals, filtered, and analyzed directly. Initial transformation of both compounds was more rapid with molasses. NPP at 100 mg liter^–1 decreased in 12 days to 50% of the initial amount and then remained constant, but PED had completely disappeared by 7 days (Fig. 3). When NPP was added at 50 mg liter^–1, all of it disappeared (Fig. 3) and the same metabolites (Table 1) were observed.

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FIG. 3. Percentages of N-phenylpiperazine or N-phenylethylenediamine remaining in cultures of WWTP reactor liquor-inoculated medium with molasses, compared to sterile controls, as shown by HPLC (A254). Cultures were dosed with NPP at 100 mg liter–1 () and 50 mg liter–1 () or with PED at 100 mg liter–1 (). Error bars represent standard errors of three samples.

Overall, when NPP was used as a model substrate, PED was a transient intermediate and N-nitroso-NPP and N-nitroso-PED were the final products. Various human-associated bacteria mediate the nitrosation of drugs (2, 3, 29); N-nitroso-norfloxacin has less antibacterial activity than norfloxacin (2). Some bacteria that remove an ethylene group from NPP may also be able to metabolize the piperazine ring of fluoroquinolones. Nevertheless, our experiments, which simplified the complexity of the WWTP environment, required an excessively long time for NPP modification. Future endeavors should include searches for bacteria that degrade specific fluoroquinolones and determination of optimal conditions for reducing environmental retention times for those fluoroquinolones.

 
We thank A. J. Williams, C. A. Elkins, B. D. Erickson, and G. Gamboa da Costa for technical advice and C. E. Cerniglia for research support. We also thank the Little Rock Wastewater Utility for allowing us to take samples.

This work was supported in part by appointments to the Postgraduate Research Fellowship Program (C.M.J.) and the Summer Student Research Program (R.S.) at the National Center for Toxicological Research, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

 
* Corresponding author. Mailing address: National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079. Phone: (870) 543-7059. Fax: (870) 543-7307. E-mail: john.sutherland{at}fda.hhs.gov

Published ahead of print on 1 August 2008.

Present address: Department of Molecular Biology and Microbiology, University of Central Florida, 12722 Research Parkway, Orlando, FL 32826.

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Applied and Environmental Microbiology, October 2008, p. 6147-6150, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00516-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jung, C. M. Articles by Sutherland, J. B. Search for Related Content PubMed PubMed Citation Articles by Jung, C. M. Articles by Sutherland, J. B. Agricola Articles by Jung, C. M. Articles by Sutherland, J. B.