Evaluation of Sulfadiazine Degradation in Three Newly Isolated Pure Bacterial Cultures

This study is aimed to assess the biodegradation of sulfadiazine (SDZ) and characterization of heavy metal resistance in three pure bacterial cultures and also their chemotactic response towards 2-aminopyrimidine. The bacterial cultures were isolated from pig manure, activated sludge and sediment samples, by enrichment technique on SDZ (6 mg L-1). Based on the 16S rRNA gene sequence analysis, the microorganisms were identified within the genera of Paracoccus, Methylobacterium and Kribbella, which were further designated as SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47. The three identified pure bacterial strains degraded up to 50.0, 55.2 and 60.0% of SDZ (5 mg L-1), respectively within 290 h. On the basis of quadrupole time-of-flight mass spectrometry and high performance liquid chromatography, 2-aminopyrimidine and 4-hydroxy-2-aminopyrimidine were identified as the main intermediates of SDZ biodegradation. These bacteria were also able to degrade the metabolite, 2-aminopyrimidine, of the SDZ. Furthermore, SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47 also showed resistance to various heavy metals like copper, cadmium, chromium, cobalt, lead, nickel and zinc. Additionally, all three bacteria exhibited positive chemotaxis towards 2-aminopyrimidine based on the drop plate method and capillary assay. The results of this study advanced our understanding about the microbial degradation of SDZ, which would be useful towards the future SDZ removal in the environment.


Introduction
Sulfadiazine [4-amino-N-(2-pyrimidinyl)benzene sulfonamide, SDZ] is one of the most common sulfonamide antibiotics, utilized widely in animal husbandry to treat and inhibit bacterial diseases [1]. Conversely, treated animals have been reported to excrete unbroken SDZ with its metabolite(s) into the ecosystem through their excretory system [2]. For example, SDZ has been detected in the range of 0.3-198 mg kg -1 (dry matter of pig manure), depending on medication, dilution and age of the manure [3,4] and also in slurry at high concentrations, almost 500 mg kg -1 [5]. Studies revealed that SDZ is mainly released into soil either by living systems and/or by application of contaminated manure from treated livestock as fertilizer on farming land [6]. Moreover, SDZ was also detected in Jiulong river and its estuary in south-east region of China [7]. The fate and effects of SDZ in manure during storage and into manure-applied soils have also been reported [8][9][10]. Hence, the persistence of SDZ in the environment is of great concern, because it might lead to severe damages to non-target organisms and thereby favors the increase of resistant bacteria and pose adverse health effects to human beings [11]. It is therefore necessary to study the metabolic fate of SDZ in the environment and its environmental degradation.
There are reports on sulfonamide antibiotics sorption activity, mobility, transformation as well as mineralization and the spread of antibiotic genes [12][13][14][15][16][17]. SDZ undergoes numerous transformation pathways and during the process can be inactivated (acetylation), altered into a less toxic molecule (hydroxylation), and/or to a highly hydrophilic metabolite with a lower molecular mass [18]. However, there are only limited reports on SDZ-degrading strains. To date, almost all independently isolated SDZ-degrading bacteria belonged to Microbacterium genus [19,20]. On the other hand, 2-aminopyrimidine (major intermediate of SDZ) was not completely mineralized by strain SDZm4 [20]. Recently, degradation of 2-aminopyrimidine by Terrabacter-like bacterium has been studied and reported [21]. However, there is not much information available on both SDZ and 2-aminopyrimidine degradation by a single pure bacterium. Therefore, it is necessary to identify microorganisms with the ability to degrade SDZ and its major metabolite, 2-aminopyrimidine. In this study, we report the isolation and characterization of three different bacteria capable of degrading SDZ as well as 2-aminopyrimidine, and their resistance capabilities towards various heavy metals. In addition, we also report for the first time positive chemotactic response of Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 and Kribbella sp. SDZ-3S-SCL47 towards 2-aminopyrimidine.

Materials and Methods
Chemicals and composition of the growth medium Sulfadiazine was purchased from Sigma-Aldrich, Saint Louis, USA. Acetone, acetonitrile and methanol were purchased from Merck, Darmstadt, Germany. All other chemicals were of analytical grade or the highest grade available commercially. Stock solutions of SDZ, 2-aminopyrimidine and 2-amino-4-hydroxypyrimidine (1.00 g L -1 ) were prepared individually using methanol and stored in amber bottles at 4°C before use. The ammonium mineral salts (AMS) medium plus yeast extract (0.04%) supplemented with substrates like SDZ (5 mg L -1 ) and 2-aminopyrimidine (5 mg L -1 ) were prepared individually by the method described previously [22,23]. The AMS medium was set to pH 7.00 (using 2 M NaOH or 2 M HCl), dispensed in 100 mL quantities into 250 mL Erlenmeyer flasks and finally sterilized by autoclaving for 20 min at 15 psi. Substrates (5 mg L -1 ) dissolved in methanol, were added to the autoclaved medium and after the evaporation of methanol, medium were inoculated.

Isolation of microorganisms by enrichment culture technique
SDZ-degrading bacteria were isolated from pig manure (PM2), activated sludge (WWTPs, W2) and sediment (3S) samples, by enrichment on SDZ (6 mg L -1 ) as a sole source of carbon and energy. PM2 and 3S were collected from Maoming, China, whereas W2 was collected from Xiamen, China (approved by Xiamen Water Affairs Zhonghuan Sewage Treatment Co. Ltd.). Samples (5 g/ 5 mL) were suspended in 100 mL of sterile distilled water, mixed and filtered. The 5 mL of filtrates were added to 95 mL of sterile AMS medium (without yeast extract) in a 250 mL of Erlenmeyer flasks supplemented with SDZ (6 mg L -1 ) as a sole source of carbon and energy. The flasks were then incubated on a rotary shaker (150 rpm) at 30°C under dark condition for 30 days. After 30 days, 5 mL of the inoculums were transferred into fresh medium containing SDZ (6 mg L -1 ) and incubated for 30 days (until turbid) and the process was repeated four more times. The bacterial consortia were purified by serial dilution and pour plate method. AMS solid medium (without yeast extract) was prepared by adding 1.65% agar. A bacterial consortium was diluted serially and cultured on SDZ (6 mg L -1 )-AMS agar plates. Different colonies obtained from bacterial consortium were further grown on AMS agar plates supplemented with SDZ (6 mg L -1 ). This process was performed several times to obtain pure bacterial cultures. The purified bacterial cultures designated as SDZ-PM2-BSH30 (PM2), SDZ-W2-SJ40 (W2) and SDZ-3S-SCL47 (3S) showed better growth on SDZ-AMS agar plates and therefore used in further studies.

16S rRNA gene sequence analysis for the identification of bacteria
For DNA, single colony of pure bacteria was transferred into 50 μL lysis buffer (Takara) in 1.5 mL tube, boiled at 80°C for 15 min and centrifuged (slowly). DNA sample (1 to 5 μL) was used for 16S rRNA gene amplification. The 16S rRNA gene was amplified by PCR using a pair of universal primers: 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTT TGTTACGACTT-3') [24]. PCR mixture contained 12.5 μL Premix Taq (Takara Biotechnology Co., Ltd, Dalian, China), 0.5 μM of each primer, approximately 1 μL of DNA template and sterile deionized water to make up the total reaction volume of 25 μL. PCR amplification was performed with a GenePro Thermal Cycler (Bioer, Hercules, China) and the amplification program consisted of: 1 cycle for 8 min at 94°C, followed by 37 cycles of 30 s at 94°C, 30 s at 55°C, 1.5 min at 72°C; and finally 1 cycle for 5 min at 72°C. The purified PCR products of 16S rRNA genes were sequenced and analyzed by comparing and aligning with relative gene sequences available in Ez-Taxon and GenBank data libraries using BLAST-n and CLUSTAL-W program. The phylogenetic trees were constructed on the basis of maximum-likelihood method using the Molecular Evolutionary Genetics Analysis software (MEGA6) [25].

Growth and degradation of SDZ and 2-aminopyrimidine
To monitor the effects of initial concentrations of SDZ and 2-aminopyrimidine on the growth of Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 as well as Kribbella sp. SDZ-3S-SCL47 and their degradation, the microorganisms were grown on 100 mL of AMSyeast extract (0.04%) in 250 mL Erlenmeyer flasks supplemented with appropriate concentration of SDZ (2-8 mg L -1 ) as well as 2-aminopyrimidine (3-7 mg L -1 ). The bacterial growth was measured at 600 nm by UV-spectrophotometer (UV-5200 Spectrophotometer). For SDZ (5 mg L -1 ) and 2-aminopyrimidine (5 mg L -1 ) degradation studies, the samples collected at regular intervals were centrifuged (7000 × g) and then analyzed by high performance liquid chromatography (HPLC). Detailed description is provided in Supporting Information. Uninoculated culture flasks with the same amount of SDZ, 2-aminopyrimidine, SDZ plus sterilized individual bacterial cells and 2-aminopyrimidine plus sterilized individual bacterial cells were served as controls.

Identification of metabolites
The metabolites of SDZ (5 mg L -1 ) in cell-free filtrates of Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 and Kribbella sp. SDZ-3S-SCL47 were identified by quadrupole time-of-flight-mass spectrometry (MS) (Q-/TOF-MS, SI). The cell-free filtrates were collected at regular intervals. The same individual bacterial cultures supernatant without SDZ were used as negative controls, and uninoculated controls containing SDZ (5 mg L -1 ) were included as well. The collected samples were centrifuged (7000 × g) for 20 min, adjusted to pH 2.0 with 2 M HCl and then extracted with ethyl acetate. The extracted samples were reconstituted in methanol (500 μl), filtered and analyzed by Q/TOF-MS.

Chemotaxis towards 2-aminopyrimidine
The chemotactic response of Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 and Kribbella sp. SDZ-3S-SCL47 towards 2-aminopyrimidine was examined qualitatively (drop plate assay) and quantitatively (capillary assay) by following procedures described earlier [27,28]. For drop plate method, the bacterial cells of SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47 were grown in LB supplemented with SDZ (5 mg L -1 ). The cultures were harvested at mid-log phase (O.D 600 between 0.60-0.70) by centrifugation at 4000 × g for 10 min and pellets were washed twice with phosphate buffered saline (PBS), resuspended in drop plate assay medium (AMS with 0.3% bacto agar) and transferred into petriplates (96 mm). Pinch of 2-aminopyrimidine was placed in the middle of plates and then incubated at 25°C. The chemotactic response was observed after 5-8 h of incubation. For quantitative capillary method, the optimum concentration of 2-aminopyrimidine was determined by carrying out at various concentrations of 2-aminopyrimidine (2-8 mg L -1 ). Initially, 10 μL of glass capillaries were filled with the desired amount of 2-aminopyrimidine (in chemotaxis buffer) and the suction end was closed by sterile agarose gel. The control capillaries were without any chemotactic compound. Capillaries were individually inserted into micro centrifuge tubes (separately) and having a suspension (10 10 to 10 11 cells mL -1 ) of SDZ-PM2-BSH30, SDZ-W2-SJ40 as well as SDZ-3S-SCL47 and were incubated at 25°C for 40 min. The solutions of capillaries were then serially diluted and spread onto LB agar plate, followed by determination of CFUs count after 48 h incubation at 37°C. Chemotaxis index (ratio of the number of CFUs produced from capillary supplemented with test substrate to CFUs produced from a control capillary) was used to quantify the chemotactic response. Aspartate was used as the positive control. No further SDZ degradation was observed even after incubating for prolonged periods (>320 h). In addition, the bacteria were also able to grow on yeast extract (0.04%)-AMS medium in the presence of 2-aminopyrimidine and 2-amino-4-hydroxypyrimidine at an initial concentration of 5 mg L -1 , respectively. Furthermore, 2-aminopyrimidine (5 mg L -1 ) was degraded up to 47.8, 57.6 and 66.4% by SDZ-PM2-BSH30, SDZ-W2-SJ40 and SDZ-3S-SCL47, respectively within 210 h (Fig 3). On the other hand, in

Identification of metabolites
The identification of SDZ metabolites was conducted by high resolution MS and HPLC. S1
Drop plate assays showed the formation of bacterial ring around the crystals of 2-aminopyrimidine after incubation between 4-6 h (Fig 5). This is well supported to the data of capillary assay.

Discussion
In this study, the screening of SDZ-degrading bacteria by enrichment method from various samples like PM2, W2 and 3S allowed to select the potential isolates with high survivability to various heavy metals and chemotactic towards 2-aminopyrimidine. Overall, three newly isolated pure bacteria were studied and compared for their degradation capability on both SDZ and 2-aminopyrimidine at 5 mg L -1 . The bacterial clutters like SDZ-W2-SJ40 and SDZ-3S-SCL47 showed higher degradation rate of SDZ (5 mg L -1 ). None of these bacteria were previously reported for the degradation of both SDZ and 2-aminopyrimidine. In addition, SDZ-W2-SJ40 and SDZ-3S-SCL47 showed higher degradation of 2-aminopyrimidine than SDZ-PM2-BSH30. There are reports on addition of yeast extract during the degradation of toxic or inhibitory pollutant(s) by pure bacteria and/or bacterial consortia [22,29]. Previous studies reported that the addition of yeast extract increases the growth of microorganisms thereby enhancing the toxic pollutant degradation [29,30]. In the present study, addition of 0.04% of yeast extract helped to enhance the bacterial growth and achieve faster degradation of SDZ, but the three strains could also degrade SDZ without addition of 0.04% of yeast extract (data not shown).
On the basis of identified metabolites and also degradation of 2-aminopyrimidine in SDZ-3S-SCL47, a pathway for the degradation of SDZ in Kribbella sp. SDZ-3S-SCL47 was proposed (Fig 6). Similarly, in other two bacteria like SDZ-PM2-BSH30 and SDZ-W2-SJ40, SDZ was also transformed into 2-aminopyrimidine which was further converted to 2-amino-4-hydroxypyrimidine (data not shown). No further metabolites were observed in all three pure bacterial cultures. Moreover, all three bacteria showed the ability to degrade 2-aminopyrimidine. The initial step of SDZ catabolism involved ipso-hydroxylation to form 2-aminopyridine whereas other by-products were not detected in this case. Similarly, in Microbacterium lacus strain SDZm4, SDZ was transformed into 2-aminopyrimidine [20]. The metabolite 2-aminopyridine was observed not only by microbial process, but also detected during photolysis, electrochemical oxidation and sorption experiments by soils [1,18,31,32]. Furthermore, in strain 2APm3, 2-aminopyrimidine was metabolized into two by-products with one of that identified as 2-amino-4-hydroxypyrimidine [21]. Similarly, in this study, the bacteria degraded 2-aminophyrimidine into 2-amino-4-hydroxypyrimidine.
In addition, Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 and Kribbella sp. SDZ-3S-SCL47 were resistant to various heavy metals. Paracoccus sp. SDZ-PM2-BSH30 showed high resistance to Pb, Methylobacterium sp. SDZ-W2-SJ40 showed high resistance to Pb, Co and Zn whereas Kribbella sp. SDZ-3S-SCL47 showed high resistance towards Pb, Ni and Zn. These high levels of heavy metal resistance could be beneficial for living and adjusting of these microbial strains in severe heavy metal polluted environment. There are reports on various organisms capable of resisting different heavy metals [26,[33][34][35][36][37]. Future study using molecular biology techniques, possibly will help to understand the mechanism of heavy metal resistance in SDZ-degrading pure bacteria.
Microbial chemotactic response was identified as an important phenomenon of bacteria to different toxic pollutants and thus, such bacteria were useful for the bioremediation of toxic pollutants in the environment [38]. Hence, it is suggested that chemotactic response can increase the biodegradation through improving the availability of substrate to support the development of mixed bacteria with the capabilities of biodegradation [27,38,39]. There are reports on various bacteria having the ability of chemotactic response towards various toxic pollutants such as nitroaromatics, organophosphate, chloroaromatics and aminobenzoates etc. [27,40,41]. In this study, SDZ-degrading bacteria such as Paracoccus sp. SDZ-PM2-BSH30, Methylobacterium sp. SDZ-W2-SJ40 and Kribbella sp. SDZ-3S-SCL47 showed chemotaxis towards 2-aminopyrimidine, which these bacteria can degrade metabolically or co-metabolically. The chemotactic response (capillary method) results in this study were similar to the results observed in Pseudomonas sp. strain BUR11 (chemotaxis towards organophosphate compound) [40]. However, it is necessary to investigate at genomic level for better understanding of this chemotactic activity in all of these three pure bacterial cultures, since this feature is a novel and characteristic to our SDZ-degrading bacteria. From previous reports, it has been observed that the bacterial chemotactic response towards various aromatic compounds proceeds via two different mechanisms (metabolism dependent and metabolism independent). There are reports on various microorganisms, which showed metabolism dependent chemotactic response to different compounds [27,42,43]. Similarly, metabolism independent chemotaxis has been also reported for different bacterial strains towards various toxic chemicals [41,44,45]. However, we are not sure whether this chemotaxis behavior is metabolically independent and/or dependent [40,42], although results presented in this study demonstrated that all three bacteria had the ability to degrade both SDZ and 2-aminopyrimidine. More experiments related to chemotaxis behavior of these bacteria for those substrates which these strains were not able to metabolize and/or transform will be needed to clarify the chemotactic mechanism.