Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics

STOTEN-21662; No of Pages 10 Science of the Total Environment xxx (2016) xxx–xxx

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Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics Diogo A.M. Alexandrino a,b,c, Ana P. Mucha a, C. Marisa R. Almeida a, Wei Gao d, Zhongjun Jia d, Maria F. Carvalho a,⁎ a Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos s/n, 4450-208 Matosinhos, Portugal b Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal c Faculty of Sciences, University of Porto, Rua do Campo Alegre 790, 4150-171 Porto, Portugal d State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, Jiangsu Province, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Rhizosediment of plants from constructed wetlands were used as microbial inocula. • The acclimation with ENR and CEF led to changes on microbial communities. • Biodegradation of ENR was never complete and defluorination was the limiting step. • CEF was always completely removed by a combination of biotic and abiotic degradation. • Concomitant presence of the two antibiotics did not influence their biodegradation.

a r t i c l e

i n f o

Article history: Received 19 October 2016 Received in revised form 19 December 2016 Accepted 20 December 2016 Available online xxxx Editor: Jay Gan Keywords: Biodegradation Enrofloxacin Ceftiofur Microbial communities Metagenomics

a b s t r a c t Fluoroquinolones and cephalosporins are two classes of veterinary antibiotics arising as pollutants of emerging concern. In this work, the microbial degradation of two representative antibiotics of both these classes, enrofloxacin (ENR) and ceftiofur (CEF), is reported. Biodegradation of the target antibiotics was investigated by supplementing the culture medium with ENR and CEF, individually and in mixture. Microbial inocula were obtained from rhizosphere sediments of plants derived from experimental constructed wetlands designed for the treatment of livestock wastewaters contaminated with trace amounts of these antibiotics. Selected microbial inocula were acclimated during a period of 5 months, where the antibiotics were supplemented every three weeks at the concentration of 1 mg L−1, using acetate as a co-substrate. After this period, the acclimated consortia were investigated for their capacity to biodegrade 2 and 3 mg L−1 of ENR and CEF. Complete removal of CEF from the inoculated culture medium was always observed within 21 days, independently of its concentration or the concomitant presence of ENR. Biodegradation of ENR decreased with the increase in its concentration in the culture medium, with defluorination percentages decreasing from ca. 65 to 4%. Ciprofloxacin and norfloxacin were detected as biodegradation intermediates of ENR in the microbial cultures supplemented with this antibiotic,

Abbreviations: CEF, ceftiofur; CIP, ciprofloxacin; CP, cephalosporin; ENR, enrofloxacin; FQ, fluoroquinolones; MM, minimal medium; NOR, norfloxacin; OD, optical density; OTU, Operational Taxonomic Unit; QIIME, Quantitative Insights into Microbial Ecology; TISAB, total ionic strength adjusting buffer. ⁎ Corresponding author at: CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos s/n, 4450-208 Matosinhos, Portugal. E-mail address: [email protected] (M.F. Carvalho).

http://dx.doi.org/10.1016/j.scitotenv.2016.12.141 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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indicating that defluorination of at least part of ENR in these cultures is not an immediate catabolic step. Abiotic mechanisms showed high influence in the removal of CEF, affecting less ENR degradation. The acclimation process with the target antibiotics led to significant shifts in the structure and diversity of the microbial communities, predominantly selecting microorganisms belonging to the phyla Proteobacteria (e.g. Achromobacter, Variovorax and Stenotrophomonas genera) and Bacteroidetes (e.g. Dysgonomonas, Flavobacterium and Chryseobacterium genera). The results presented in this study indicate that biodegradation can be an important mechanism for the environmental removal of the tested compounds. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Veterinary drugs are commonly used to treat numerous animal diseases. Antibiotics constitute one of the most broadly applied groups of these pharmaceuticals, being used not only for the treatment and prevention of diseases, but also for the promotion of animal growth and improvement of the nutritional value of animal-based foodstuffs, despite the legal restrictions concerning these latter applications (Cromwell, 2002; Li et al., 2011). The overuse of veterinary drugs has contributed to the emergence of these products in several environmental compartments, essentially as a result of the employment of contaminated livestock waste as natural fertilizers (Loke et al., 2000; Tasho and Cho, 2016). In addition, these drugs are also released to the environment through wastewater treatment plants (WWTP) effluents, because WWTP are, in most cases, not capable of dealing with this type of contaminants, resulting in incomplete or even no removal of these compounds from agro-industrial effluents (Corcoran et al., 2010). Pharmaceuticals may be released to the environment in their parental form or as metabolites, which may still hold biological activity. As these compounds are designed to induce specific physiological and biochemical effects on target organisms, their environmental presence can cause a wide range of toxic effects (Sarmah et al., 2006). For the particular case of antibiotics, their environmental presence may also promote the selection of antibiotic-resistant microorganisms (Martinez, 2009). Fluoroquinolones (FQ) and cephalosporins (CP) are two of the most widely used antibacterial pharmaceuticals worldwide. In 2012, the consumption in Europe of both FQ and CP accounted for over 20% of the total antibiotics consumption (Weist et al., 2014). FQ are piperazinyl derivatives of the N-heterocyclic antibacterial compounds designated as quinolones (Felczak et al., 2014). Their mode of action relies on the ability to inhibit the activity of topoisomerases type II and IV, key enzymes in DNA replication, which leads to the blockage of microbial cell multiplication (Hu et al., 2007). CP are semi-synthetic analogous of the naturally-produced cephalosporin-C (Rex and Susan, 2002). Being a class of β-lactam antibiotics, their antibacterial activity resides in their capability to disrupt peptidoglycan biosynthesis, affecting bacterial-cell integrity (Rex and Susan, 2002). Both classes of antibiotics have a broadspectrum activity towards several aerobic and anaerobic pathogens. FQ have been widely reported to occur in both terrestrial and aquatic ecosystems in trace concentrations, typically ranging from ng L−1 to μg L−1, though concentrations of several mgL−1 have also been reported (Picó and Andreu, 2006; Larsson et al., 2007; Zhang and Li, 2011). Physical-chemical properties of CP promote a faster environmental dissipation of these antibiotics, leading to lower residence times of these pharmaceuticals in the environment (Junker et al., 2006) and lower detections. As a consequence of the environmental release of these two classes of antibiotics, an increasing number of microorganisms resistant to these drugs has been reported in the literature (Miranda and Castillo, 1998; Walsh, 2000; Ho et al., 2001; Hooper, 2002; Su et al., 2008), highlighting the importance of studying their biodegradation potential. Microorganisms play a central role in the biodegradation of environmental pollutants, including pharmaceutical compounds. The presence of environmental contaminants frequently leads to shifts on functional diversity, abundance and organization of microbial communities colonizing affected areas, which may compromise ecosystems equilibrium.

Metagenomics approaches have been employed as a powerful tool to unveil how microbial communities adapt and respond to environmental contaminants, allowing better understanding their impact on microbial ecology (Röling and van Bodegom, 2014). The main objective of this work was to investigate the capacity of microbial communities from experimental constructed wetlands to biodegrade two antibiotics representative of the FQ and CP groups, enrofloxacin (ENR) and ceftiofur (CEF), respectively. ENR has been reported to occur in wastewaters, agricultural soils and animal manure, while several metabolites of CEF resultant from animal detoxification have been detected in manure and soils (Rex and Susan, 2002; Zhao et al., 2010; Sim et al., 2011; Li et al., 2014). Also, ENR and CEF may occur simultaneously in the environment, due to their similar prophylaxis and its frequent use in veterinary applications. Degradation of these compounds mainly focuses in physical-chemical processes (Sturini et al., 2012; He et al., 2014; Zamanpour and Mehrabani-Zeinabad, 2014; Yang et al., 2016), while less studies are found in the literature concerning their biodegradation (Martens et al., 1996; Wetzstein et al., 1997; Rafii et al., 2009; Erickson et al., 2014). In the present work, biodegradation of ENR and CEF was investigated individually and in mixture in order to understand if their biological removal was affected by their concomitant presence. Moreover, the effect of these antibiotics in the microbial dynamics of the degrading cultures was studied through metagenomics analysis. 2. Materials and methods 2.1. Reagents The quality of all chemicals used was pro analisis or equivalent. ENR, CEF, ciprofloxacin (CIP) and norfloxacin (NOR) were purchased from Sigma-Aldrich® (Barcelona, Spain). Working solutions of the antibiotics were prepared in methanol at a concentration of 1 g L−1 and stored at −20 °C. Methanol and formic acid were acquired from Sigma-Aldrich® (Barcelona, Spain). 2.2. Acclimation of microbial degrading cultures Microbial cultures capable of degrading ENR and CEF were obtained by acclimation of inoculated cultures with the target antibiotics, supplemented either individually or in mixture, and using acetate as a co-substrate. Rhizosphere sediment samples obtained from experimental constructed wetlands were used as inocula. These experimental systems were planted with Phragmites australis (in a layered substrate composed of gravel, lava rock and plants roots bed substrate) and designed for the treatment of pig farm wastewaters, which were artificially contaminated with ENR or CEF (100 μg L−1) either individually or in a mixture (Almeida et al., 2017). The rhizosphere sediment samples used as inocula were collected from each of these systems after 20 weeks of operation. The process of acclimation was conducted in duplicate, in batch mode and under aerobic conditions, along a period of 5 months. For that, 250 mL flasks containing 50 mL of sterile minimal salts medium (MM) were inoculated with 5 g of rhizosphere sediment, which was obtained from one of the three experimental constructed wetlands systems, i.e., constructed wetlands supplemented with pig farm wastewaters doped with ENR, doped with CEF or doped with a mixture

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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of both antibiotics. The flasks were fed with the respective antibiotics at a concentration of 1 mg L− 1 and acetate at a concentration of 400 mg L−1. To ensure sterile conditions, the flasks were kept closed throughout the experiment (Fig. 1S). MM contained (per liter): Na2HPO4·2H2O 2.7 g, KH2PO4 1.4 g, (NH4)2SO4 0.5 g, MgSO4·7 H2O 0.2 g and 10 mL of a trace elements solution with the following composition, per liter: Na2EDTA·2H2O 12.0 g, NaOH 2.0 g, MnSO4·4H2O 0.4 g, ZnSO4·7H2O 0.4 g, H2SO4 0.5 mL, Na2SO4 10.0 g, Na2MoO4·2 H2O 0.1 g, FeSO4·7H2O 2.0 g, CuSO4·5H2O 0.1 g and CaCl2 1.0 g. Microbial cultures were incubated in a rotary shaker (Selecta model 2102, JP Selecta S. A., Barcelona, Spain, 130 rpm), at 25 °C and protected from light. Acetate was fed to the cultures twice a week. Every 3 weeks, 25 mL of the microbial cultures were transferred to new flasks containing equal volume of MM and re-fed with the target antibiotics and acetate, thus initiating a new feeding cycle. Every week, cultures were transferred to new flasks to assure appropriate aerobic conditions. For each feeding cycle of the acclimation phase, microbial degradation was followed by monitoring microbial growth (every 7 days), fluoride ion release for ENR (every 7 days) and by measuring the concentration of ENR and CEF in the culture medium (at the end of the feeding cycle). 2.3. Biodegradation of different concentrations of ENR and CEF After the acclimation period, biodegradation of the target antibiotics was investigated for the concentrations of 2 and 3 mg L−1. The experimental approach used was similar to the acclimation phase. For the biodegradation experiments, microbial cultures acclimated in the previous phase were diluted to half, in order to obtain a final culture volume of 50 mL. The experiments were conducted in triplicate using acetate as a co-substrate, testing firstly the concentration of 3 mg L−1 followed by 2 mg L−1. Antibiotics were supplemented to the cultures in these concentrations in three week intervals, for a total of two feeding cycles. Biodegradation was monitored by analyzing at the end of each feeding cycle the same analytical parameters indicated in the acclimation phase. In parallel with the biodegradation experiments, two sets of abiotic controls were established in order to investigate the influence of abiotic mechanisms in the removal of the target antibiotics. Abiotic controls were established using an antibiotics concentration of 2 mg L−1 as it corresponded to an intermediate concentration tested in the experiments. One abiotic control set consisted in sterile MM supplemented with ENR or CEF, either individually or in mixture. The other abiotic control set consisted in sterile MM inoculated with autoclaved microbial consortia obtained from the acclimation phase (initial optical density at 600 nm of 0.1), supplemented with the target antibiotics. Abiotic controls were established in triplicates and incubated for one month in the same conditions of the biodegradation experiments. 2.4. Analytical methods Biomass growth was monitored by reading the absorbance of culture samples at 600 nm, in a spectrophotometer (V-1200, VWR International, USA). Fluoride ion release was measured as an indicator of ENR defluorination. The concentration of fluoride ion in solution was analyzed, after centrifuging samples at 13000 rpm for 15 min (VWR Microstar 17, VWR International, USA), with a combination fluoride-selective electrode (Crison 9655 C, Crison Instruments, S.A., Spain). Prior to sample analysis a calibration curve was obtained using standard solutions of sodium fluoride (0.001 to 1 mM) prepared in MM. A total ionic strength adjustment buffer (TISAB III) was supplemented to the samples and standards in a 1:10 ratio. CEF, ENR and its intermediates CIP and NOR were analyzed in the supernatant of the culture samples by HPLC. Supernatants were obtained through centrifugation at 13000 rpm for 15 min. Separation of the target antibiotics was performed in a C18 Luna column (150 × 4.6 mm) from Phenomenex, coupled to a Beckman Coulter HPLC equipped with a

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diode array detector (module 128) and an automatic sampler (module 508). Chromatographic conditions were the same as those optimized by Cavenati et al. (2012). Briefly, water-formic acid (99.9:0.1, v/v; eluent A) and acetonitrile (eluent B) were used as eluents. Isocratic conditions (100% of eluent A) were maintained in the first 2 min followed by a 10 min linear gradient (70% of eluent A and 30% of eluent B), after which the initial isocratic conditions were reached again for an additional 2 min. Flow rate was 1.0 mL min−1 in the isocratic steps of the run and lowered to 0.8 mL min−1 in the linear gradient step. The sample injection volume was 50 μL. ENR, CIP and NOR were screened at 280 nm, while CEF was detected at 290 nm. Analyses were carried out at room temperature (ca. 20 °C). The analytical detection limit (LOD) for all the target antibiotics was 0.1 mgL−1. Standard solutions of the antibiotics were prepared in MM (0.1–6 mg L−1) and used to obtain calibration curves prior to every analysis. 2.5. Analysis of the structure of the microbial communities The effect of the acclimation process with the target antibiotics in the degrading cultures, was investigated by comparing the structure of the different microbial communities. For that, the initial inocula (rhizosphere sediment samples collected from each of the three constructed wetlands systems) was compared with that of the respective microbial cultures obtained at the end of the biodegradation experiments. Due to the fact that the initial sediment samples and the final degrading cultures constitute two very distinct matrixes, different DNA extraction methods were employed in order to obtain optimal DNA yield and quality. DNA from the sediment samples used as inocula for the experiments was extracted from 0.5 g (wet weight) of homogenized sediment using PowerSoil® DNA Isolation Kit from MOBIO Laboratories, Inc., according to the manufacturer's instructions. DNA from the degrading cultures was obtained using a standard phenol-chloroform extraction method, as described elsewhere (Sambrook et al., 1989). For each treatment, DNA samples were extracted in duplicate and then pooled as composite samples for metagenomics analysis. Structure of microbial communities in different samples was assessed by Illumina Miseq sequencing of the 16S rRNA gene. Fusion primers consisting of adaptor A or B, key sequence, barcode and template specific sequences were used in this study. Specifically, the V4V5 region of the bacterial 16S rRNA gene was amplified by Polymerase Chain Reaction (PCR) with the forward primer 515F (5′GTGCCAGCMGCCGCGG-3′) and the reverse primer 907R (5′CCGTCAATTCMTTTRAGTTT-3′), and a 12 bp adaptor sequence was attached to the 5′ end of 515F (Xia et al., 2011). The 50 μL PCR reaction mixture contained 1 × PCR buffer (Mg2 + plus), 0.2 mM of each deoxynucleoside triphosphate, 0.4 mM of each primer, 1.25 U of TaKaRa Taq HS polymerase (TaKaRa Biotech, Dalian, China) and 1 μL template DNA. The PCR amplification program included initial denaturation at 94 °C for 5 min, followed by 32 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 5 min. Amplified products were subjected to electrophoresis using a 1.8% agarose gel. Amplicon bands with a suitable size (475 bp) were excised from the gel using a clean and sharp scalpel under ultraviolet illumination. The residual buffer was then discarded before the excised gel slice was placed into a centrifugation tube for DNA purification with an agarose gel DNA purification kit (TaKaRa Biotech, Dalian, China). All of the purified amplicons were then combined in equimolar amounts and submitted to high-throughput sequencing on an Illumina MiSeq pyrosequencer. The MiSeq sequencing data was analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline (http:// qiime.org/) (Kuczynski et al., 2011). Briefly, low quality sequences, which have lengths of b 200 bp, an average quality score of b25 and primer mismatches were trimmed and the barcodes were determined to assign sequence reads to the proper samples. Then, the chimeras were detected using the UPARSE algorithm based on a database of chimera-free sequences. The sequences, which were assigned to a

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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mitochondrial or chloroplast origin were eliminated with the Metaxa software tool and the V4–V5 region was extracted with the V-Xtractor software tool. Next, all of the sequences were clustered into Operational Taxonomic Units (OTUs) at 97% sequence identity with the UCLUST algorithm and a representative sequence from each OTU was picked for taxonomic identification using Ribosomal Database Project (RDP) classifier (http://rdp.cme.msu.edu/). Bacterial community diversity was examined by rarefaction curves, non-metric multi-dimensional scaling (NMDS) and two indices: Chao1 index (Chao, 1984), which is a nonparametric index of species richness especially suitable when many low abundance classes exist, and the Shannon ‘H’ index (Shannon, 1948), which is a measure of diversity combining both abundance and evenness. The Chao1 index was calculated as Chao1 = Sobs + (n21 / 2n2) where Sobs is the number of species (OTUs), n1 the number of species represented by one individual (singletons) and n2 the number of doubletons. The results of Bray-Curtis distances were visualized by non-metric multidimensional scaling (NMDS) plots in the R environment using the ‘metaMDS’ function of the vegan package. 2.6. Statistical analysis For the biodegradation experiments, replicates of samples were analyzed independently and mean values and corresponding standard deviations were calculated. Statistical analysis was performed using the software STATISTICA version 12 (StatSoft, Inc., 2013). For antibiotic removals and ENR defluorination obtained in the biodegradation experiments with 2 and 3 mg L−1, statistically significant differences were evaluated through a parametric Student's t-test, using mean values and corresponding standard deviations of the replicates. Statistical significance was assumed when the p-value was below or equal to 0.05. 3. Results 3.1. Biodegradation of ENR and CEF To investigate the biodegradation of ENR and CEF, an acclimation period of 5 months was established using sediment samples obtained from experimental constructed wetlands treating livestock wastewaters contaminated with these antibiotics. Due to the low concentrations of ENR and CEF tested in this study, acetate was added to the cultures as a growth supporting substrate. The purpose of this acclimation phase was to allow the adaption of the microbial communities to each antibiotic. During the first nine weeks of the period of acclimation, both microbial growth and fluoride release were not analyzed in the cultures due to the interference of the sediment used as inocula in the analysis of these parameters. According to Table 1, nine weeks after the beginning of the acclimation phase, biodegradation of ENR (based on fluoride release) in the cultures fed individually with this compound and in mixture with CEF was 53% and 65%, respectively, at the end of the feeding period. In these microbial cultures, ENR was gradually defluorinated along each feeding period of 3 weeks. These results remained very similar until the end of the acclimation period (data not shown), and the complete defluorination of ENR was never achieved. During this phase, CEF was found to be always completely removed from the culture media, while ENR removals ranged between 45 and 55% when supplemented individually or in a mixture, respectively (data not shown). Along the acclimation phase, microbial cultures always had an increase on their microbial densities (supported by the addition of acetate), showing a gradual increment over time in their optical density (OD) (data not shown). After the acclimation period, microbial cultures were tested for their capacity to degrade ENR and CEF at the concentrations of 2 and 3 mg L−1. Microbial cultures were initially fed with the highest concentration, 3 mg L−1, to test their robustness to degrade the target

Table 1 Defluorination performance along a feeding period of 21 days, obtained nine weeks after the beginning of the acclimation phase, for ENR supplied individually and in mixture with CEF, at the concentration of 1 mg L−1. Time (days)

7 14 21 a

% of ENR defluorination ENRa

ENR + CEFa

18 ± 1 24 ± 6 53 ± 2

6±3 46 ± 5 65 ± 3

Results are expressed as the mean of duplicates ± standard deviation.

antibiotics. In these conditions, defluorination of ENR sharply decreased, being obtained values of ca. 4 and 3% in the cultures fed with ENR and with a mixture of ENR and CEF, respectively (Fig. 1). However, based on antibiotics analysis in the supernatant of culture medium, these microbial cultures were able to consume ca. 40% of the supplemented ENR (Fig. 1). Removal efficiencies of 100% were always observed for CEF, both in the cultures supplemented individually with this antibiotic and in the cultures fed concomitantly with ENR (Fig. 1). When the cultures were fed with 2 mg L−1 of the target antibiotics, ENR biodegradation performance improved, namely its defluorination, despite the attained values being far below those obtained during the acclimation phase with 1 mg L− 1. Under these circumstances, similar (p N 0.05) defluorination efficiencies of ENR were achieved in the cultures fed with ENR and with a mixture of the two antibiotics, with values of 22 and 16% of defluorination being obtained, respectively. At this concentration, ENR removals were fairly constant, showing no significant differences to the ones obtained when this antibiotic was fed at 3 mg L−1 (Fig. 1). Removal efficiencies of 100% were again observed for CEF, showing no significant differences in function of its concentration or the concomitant presence of ENR (Fig. 1). In addition, for each antibiotic concentration, defluorination and removal percentages for ENR and CEF were very similar in the two feeding cycles tested. The increase in antibiotics concentrations did not affect microbial growth, being achieved OD increments similar to the ones observed in the acclimation phase (data not shown). Analyses of the antibiotics in the supernatant of the microbial cultures supplemented with 2 or 3 mg L−1 of ENR (both individually and in mixture with CEF) revealed the presence of two metabolites, though in concentrations below the limit of quantification, identified as CIP and NOR by comparison with the corresponding standard solutions.

Fig. 1. Biodegradation of ENR and CEF obtained at the end of the second feeding cycle, when supplied individually or in a mixture for the concentrations of 3 and 2 mg L−1. Results are expressed as the mean of triplicates and error bars are relative to standard deviation.

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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Comparing total removal with abiotic controls (Fig. 2), it was observed that a substantial amount of CEF was removed abiotically, having also a considerable capacity to adsorb to microbial cells. After 30 days of incubation, ca. 39% of CEF was removed in the controls with no cells, while up to 37% of this antibiotic was removed in the controls containing autoclaved consortia (Fig. 2). In contrast, ENR showed no removal or defluorination in the controls without cells, having only a slight tendency for cell adsorption, as evidenced by the ca. 6 and 9% removals obtained in the controls with autoclaved cultures supplied with ENR individually and in mixture with CEF, respectively (Fig. 2). The adsorption behavior of both antibiotics did not seem to be influenced by their simultaneous presence, as no significant differences were observed in this condition (Fig. 2).

3.2. Analysis of microbial communities' dynamics To investigate the effect of the microbial acclimation with the antibiotics, microbial compositions at the beginning (i.e., when the acclimation phase was initiated) and at the end of the experiments (i.e., when the biodegradation experiments were concluded) were compared by metagenomics analysis. A total of 178,658 sequence reads were obtained, from which 170,123 corresponded to high quality sequences. The amount of valid reads retrieved from the initial communities ranged between ca. 20,000 to 29,000 sequence reads, whereas for the acclimated consortia higher reads were obtained, varying between 27,000 and 39,000 (Table 1S). For the comparison of microbial community richness and diversity, the number of valid sequences was normalized to 20,400 reads. Clear shifts on microbial dynamics occurred as a result of the microbial acclimation with the target antibiotics, showed by the lower microbial diversity and richness verified in the degrading microbial consortia (Fig. 3). This trend is further supported by the clear dissimilarities between the structure of the acclimated communities and the initial ones, exhibited by the NMDS plot (Fig. 4). Also, the presence of the antibiotics individually or as a mixture did not seem to influence differently the structure of the acclimated microbial communities (Fig. 4). Concerning microbial structure, five dominant phyla were found in the initial communities: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Chloroflexi, accounting for over 80% of the structure of the communities of the initial inocula (Fig. 5). Microbial acclimation with the target antibiotics caused a clear decrease in the abundance of microorganisms belonging to the phyla Firmicutes and Actinobacteria, while the abundance of Proteobacteria and Bacteroidetes increased. The latter phyla represented between 80 and 90% of the entire microbial communities in the final consortia (Fig. 5). Cultures acclimated with

Fig. 3. Bacterial richness (Fig. 3A and B) and diversity (Fig. 3C) of the microbial communities at the beginning and at the end of the experiments. A – Observed OTUs; B – Chao1 estimator; C – Shannon index.

Fig. 2. Removals of ENR and CEF obtained in the biodegradation experiments (at the end of the second feeding cycle) and in the abiotic controls, for the concentration of 2 mg L−1. Results are expressed as the mean of triplicates and error bars show standard deviation.

ENR and with a mixture of ENR and CEF also showed an increase in microorganisms belonging to the phylum Spirochaetae (Fig. 5). Table 2 shows the relative abundance of the most represented taxonomic groups identified in the initial microbial communities and in the microbial cultures acclimated with the target antibiotics. The acclimation process with ENR and CEF supplied individually and in a mixture,

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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Fig. 4. Non-metric multidimensional scaling plot showing the Bray-Curtis distances among the microbial communities at the beginning and at the end of the experiments.

led to the selection of microorganisms belonging to the taxonomic groups Rhizobiales, Betaproteobacteria and Comamonadaceae and to the loss of Acidomicrobiales, Anaerolineaceae and Xanthomonadaceae (Table 2). The occurrence of the bacterial genus Dysgonomonas increased with the process of acclimation, while the genus Clostridium lost representation in all the final microbial communities (Table 2). The Betaproteobacteria class was the most representative group in the acclimated microbial communities, with the highest number of unidentified species (ranging from 33.7 to 36.5%). Despite the general shifts observed at the genus level, for all the acclimated microbial communities, metagenomics analysis showed that the mode of antibiotics supplementation led to the selection of specific genera. For the cultures acclimated with ENR, the selection of the genera Flavobacterium (20.8%) and Achromobacter (8.4%) was observed, while the genera Stenotrophomonas (12.8%) and Chryseobacterium (29.3%) increased their representation in the microbial cultures acclimated with CEF and with a mixture of ENR and CEF, respectively (Table 2). 4. Discussion There are several physical-chemical processes capable of removing FQ and CP from environmental matrices, but only a few biotic mechanisms have been described for their degradation (Sturini et al., 2012; He et al., 2014; Karlesa et al., 2014; Yang et al., 2016). The potential of environmental microorganisms to biodegrade these antibiotics is yet 100% 90%

Relative abundance

80% 70% 60% 50% 40% 30% 20% 10% 0%

ENRinitial Proteobacteria Chloroflexi Spirochaetae

CEFinitial

ENR+CEFinitial

Firmicutes Planctomycetes Other

ENRfinal

CEFfinal

Actinobacteria Deinococcus-Thermus

ENR+CEFfinal

Bacteroidetes Acidobacteria

Fig. 5. Relative abundance of the different bacterial phyla at the beginning and at the end of the experiments.

to be properly elucidated and the work developed in this study intends to shed some more light in this respect. As biodegradation by microbial communities is usually more effective than by pure cultures, this work focused on the biodegradation of ENR and CEF by microbial consortia. Biodegradation was tested for a range of concentrations between 1 and 3 mg L−1. Though these concentrations are above the ones typically found in the environment, they can reflect scenarios of acute contamination caused, for instance, by the discharge of effluents of pharmaceutical or livestock industries (Larsson et al., 2007). In this study, the acclimation phase certainly had a major role in the biodegradation performance of the degrading consortia, as it constitutes an important process in the biodegradation of environmental pollutants. This is supported by the observed shifts in the diversity and richness of the microbial communities after a prolonged time of acclimation. Liao et al. (2016) verified this when compared the biodegradation performances of CIP by non-acclimated and acclimated microbial communities, showing that this antibiotic was more readily removed by acclimated consortia. Biodegradation of ENR was investigated taking in consideration not only its removal, but also the release of fluoride ion that is usually a limiting step in the biodegradation of fluorinated compounds (Kiel and Engesser, 2015), and which has not been addressed before in studies of biodegradation of this antibiotic. ENR was shown in this study to be metabolized by the acclimated consortia, though complete defluorination and removal of this antibiotic has never been achieved. Microbial defluorination of this antibiotic was significantly influenced by its concentration, with defluorination being higher when the antibiotic was supplemented at 1 mg L−1 and declining markedly with the increase of ENR concentration. However, under these circumstances, the removal efficiency of ENR did not change significantly. This shows that the microbial cultures more readily removed ENR from the culture medium than cleaved the C\\F bond of the molecule, which is necessary for its complete degradation, thus suggesting that fluoride release constitutes a limiting step in the biodegradation of this compound. In the cultures fed with ENR (both individually and in mixture with CEF), the metabolites CIP and NOR were consistently detected, but it remained unclear if their production was a consecutive event or if it corresponded to independent metabolic pathways. Nonetheless, the identification of these metabolites in the cultures supplemented with ENR suggests that, at least, part of the molecule is not immediately subjected to an initial defluorination step, as the identified metabolites (NOR and CIP) also bear a fluorine atom in their structures. The metabolite CIP has been reported before to be involved in the biodegradation of ENR by fungal species, being produced by deethylation of the ENR piperazine ring (Wetzstein et al., 2006). On the other hand, to the best of our knowledge, NOR has never been reported before as an intermediary metabolite of ENR biodegradation. Further biodegradation of these two fluorinated metabolites are described to proceed via attack to the piperazine ring, with fluoride removal occurring afterwards through a hydroxylation reaction (Amorim et al., 2013; Liao et al., 2016). In this study, it is possible that biodegradation of ENR follows a similar pathway, in which the following steps may occur: (i) initial conversion of ENR into CIP and/or NOR; (ii) loss of the piperazine moiety in both CIP and NOR, resulting in other metabolites still bearing fluorine in their structure; (iii) defluorination of these fluorinated products by hydroxylation. This chain of reactions is expected to generate smaller and simpler molecules, with less antibacterial activity that may more easily be used as carbon sources by environmental microorganisms (Wetzstein et al., 2009; Liao et al., 2016). While defluorination of ENR may not be an immediate catabolic step, it may contribute to the inactivation of its bactericidal properties, as it has been shown before for other FQ (Carvalho et al., 2016). Studies on the biodegradation of ENR indicate that this antibiotic is degraded by various fungal species (though in different extents), with no reports on its bacterial degradation being found in the literature.

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

D.A.M. Alexandrino et al. / Science of the Total Environment xxx (2016) xxx–xxx

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Table 2 Metagenomics profiles of the initial inocula and acclimated consortia, showing the relative abundance of each taxonomic group in the communities (relative abundances below 2% were not considered).

Phylum

Class

Order

Family

Genus

Actinobacteria

Acidimicrobiia

Acidimicrobiales

Bacteroidetes

Bacteroidia Flavobacteriia

ENRinitial CEFinitial ENR+CEFinitial ENRfinal CEFfinal ENR+CEFfinal

Bacteroidales

Porphyromonadaceae

Dysgonomonas

Flavobacteriales

Flavobacteriaceae Flavobacterium Chryseobacterium

Chloroflexi

Anaerolineae

Anaerolineales

Anaerolineaceae

Deinococcus-Thermus Deinococci

Deinococcales

Trueperaceae

Firmicutes

Clostridiales

Christensenellaceae

Clostridia

Truepera

Clostridiaceae

Clostridium

Peptostreptococcaceae Proteobacteria

Erysipelotrichia

Erysipelotrichales

Alphaproteobacteria

Rhizobiales

Erysipelotrichaceae

Turicibacter

Bradyrhizobiaceae

Bosea

Brucellaceae Phyllobacteriaceae

Mesorhizobium

Xanthobacteraceae Betaproteobacteria Burkholderiales

Alcaligenaceae

Achromobacter

Comamonadaceae Variovorax Gammaproteobacteria

Neisseriales

Neisseriaceae

Xanthomonadales

Xanthomonadaceae Arenimonas Stenotrophomonas

Spirochaetae

Spirochaetes

0%

Spirochaetales

5%

10%

Spirochaetaceae

15%

20%

Different strains of Gloeophyllum striatum were reported to metabolize 5 and 10 mg L−1 of ENR, but complete degradation has never been achieved in a period of eight weeks. A small portion of the produced metabolites corresponded to non-fluorinated congeners of the parental compound, that were generated as a primary metabolic step through a hydroxylation reaction (Martens et al., 1996; Wetzstein et al., 1997; Karl et al., 2006; Wetzstein et al., 2006). On the other hand, Mucor rammannianus obtained from a forest mushroom was able to degrade ca. 79 mg L−1 of ENR in a 21 days period, though no information on defluorination of the molecule was given in the study (Parshikov et al., 2000). Contrary to what happens with ENR, the bacterial degradation of other FQ, such as CIP, NOR, moxifloxacin, etc., has been described before (Amorim et al., 2013; Maia et al., 2014; Carvalho et al., 2016; Liao et al., 2016). The enzymatic mechanisms associated with the biodegradation of these compounds are not well known, despite constituting important determinants in the antimicrobial resistance to this class of antibiotics. It has been shown that some fungal species produce laccases and peroxidases during the metabolism of FQ, including ENR, but it is not known whether these same enzymes could be responsible for the degradation of FQ by bacteria (Wetzstein et al., 2006; Prieto et al., 2011). Microbial cultures supplemented with CEF were always capable of completely removing this compound from the culture medium, independently of its concentration or the concomitant presence of ENR. Although a part of this removal was due to abiotic processes, these results are in agreement with other literature studies on the biodegradation of CEF. A wide group of anaerobic bacterial strains obtained from bovine waste were shown to be able to fully remove 5 mg L−1 of this antibiotic within 24 to 120 h (Rafii et al., 2009). Biodegradation of 10 mg L−1 of CEF by fecal microorganisms has also been reported (Li

Spirochaeta

25%

30%

35%

40%

et al., 2011; Erickson et al., 2014). Among these microorganisms, a Bacillus cereus was capable of growing with concentrations of this antibiotic above 100 mg L−1 (Erickson et al., 2014). Some of these microorganisms were found to be capable of expressing β-lactamases, a group of enzymes that play a fundamental role in the complete degradation of CEF (Rafii et al., 2009; Erickson et al., 2014). It is possible that part of the removal of CEF obtained in this work is a result of similar enzymatic activities, as the expression of β-lactamases in environmental microorganisms is a very common phenotype (Rafii et al., 2009; Bush and Jacoby, 2010; Erickson et al., 2014). It is reported that one of the primary targets in CEF biodegradation is the β-lactam moiety, a mechanism that may also have occurred in CEF degradation by the microbial consortia acclimated in this work (Li et al., 2011). This reaction may be responsible for a considerable reduction of CEF antibacterial properties, as the antibiotic potential of CP rely heavily on the integrity of their lactam ring (Rex and Susan, 2002). In the microbial cultures supplemented simultaneously with ENR and CEF, biodegradation performances of these compounds were very similar to the ones obtained in the cultures fed individually with these antibiotics. This indicates that the metabolic mechanisms responsible for CEF removal do not affect ENR degradation or vice-versa, and that the enzymes responsible for the metabolism of these two drugs are likely to be distinct. This result is highly relevant, as it suggests that the concomitant environmental presence of these two antibiotics will not hinder their microbial removal. However, it should be noted that the fate and dynamics of these antimicrobials in the environment can be greatly influenced by numerous environmental factors that can limit the biodegradation process. Both biotic and abiotic mechanisms played an important role in the removal of CEF. This is also expected to occur in an environmental

Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141

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D.A.M. Alexandrino et al. / Science of the Total Environment xxx (2016) xxx–xxx

scenario, which might explain why CP do not tend to persist in the environment. Two abiotic mechanisms, namely hydrolysis and photolysis, have been reported to be involved in the breakdown of CP, including CEF (Jiang et al., 2010; Li et al., 2011). In this work, abiotic degradation of CEF might have occurred through a hydrolysis mechanism, as the experiments were always conducted in the absence of light. Unlike CEF, abiotic degradation of ENR was found to have a minor role in the removal of this antibiotic, indicating that it was primarily degraded through the catabolic action of the acclimated consortia. In addition, abiotic controls with autoclaved consortia also showed that both ENR and CEF tended to bind to microbial membranes, with CEF showing a higher potential. While this may account as a removal mechanism, adsorbed antibiotics may still have been metabolized in the degradation experiments, as adsorption is usually a reversible reaction. The process of acclimation with the target antibiotics, supplied individually or in mixture, had a significant effect on the structure and diversity of the microbial communities. Both the individual and simultaneous presence of ENR and CEF is expected to promote microbial selection in the communities, selecting those microorganisms capable of breaking down these compounds. In this work, the acclimation process with the target antibiotics had a significant effect in the structure of the acclimated microbial communities. FQ have shown to be able to greatly influence microbial composition, but not so much diversity and richness. For example, significant shifts at the microbial community level have been associated with CIP, but diversity and richness were shown to remain largely unaltered (Girardi et al., 2011; Liao et al., 2016). Also ENR was shown before to affect microbial community structure, but significant long-term changes in microbial diversity and richness were not verified (Fernandes et al., 2015). In our work we found that ENR greatly influenced both microbial community structure and diversity. This may be a consequence of microbial cultures being exposed to antibiotics concentrations and growth conditions different than those of the experimental systems from where the inoculum samples were obtained. Overall, two of the most dominant bacterial phyla present in the initial communities, Firmicutes and Actinobacteria, suffered a considerable decrease in the acclimated consortia, with the phyla Proteobacteria and Bacteroidetes, gaining a higher expression in the acclimated communities. In a metagenomics study conducted with CIP, microorganisms belonging to Proteobacteria and Actinobacteria phyla were mainly selected, whereas Bacteroidetes and Firmicutes species lost their representation (Liao et al., 2016). The fact that in both studies a selection of microorganisms belonging to the phylum Proteobacteria, was promoted, with a special emphasis on Betaproteobacteria, suggests that members of this taxonomic group may have an important role in the biodegradation of FQ. Among the phylum Bacteroidetes, representation of the genus Dysgonomonas increased in the consortia acclimated with the target antibiotics, both individually and in mixture, indicating that this taxonomic group likely has a role in the biodegradation of both ENR and CEF. Liao et al. (2016) also found an increase of Dysgonomonas species in CIP-acclimated communities, suggesting that microorganisms belonging to this genus may be involved in the biodegradation of FQ. Other bacterial genera selected in the acclimated communities were Flavobacterium, Chryseobacterium, Achromobacter, Variovorax and Stenotrophomonas. These genera have already been associated with the biodegradation of recalcitrant organic compounds, many of them halogenated (Lo et al., 1998; Li et al., 2009; Horemans et al., 2013; Jadhav and David, 2016; Pan et al., 2016). The bacterial genera selected in this study, as a result of antibiotics acclimation, have been associated with environmental matrices similar to those from which our inocula were derived. For example, bacterial species belonging to the Flavobacterium, Chryseobacterium, Achromobacter, Variovorax and Stenotrophomonas genera have been isolated from soil, rhizosphere and wetland environments (Berg et al., 1999; Shen et al., 2005; Yoon et al., 2006; Wan et al., 2007; Miwa et al., 2008; Sang et al., 2013; Lv et al., 2014). Some Dysgonomonas species

were isolated from animal gut, suggesting that in our study they were originated from the livestock wastewaters (Pramono et al., 2015). The results obtained in this study allowed the identification of several bacterial genera that may have an important role in the biodegradation of the target antibiotics, ENR and CEF, which to our knowledge, has not been reported before. 5. Conclusion In this study, ENR and CEF were degraded at different extents by microbial communities derived from experimental constructed wetlands designed to treat wastewaters contaminated with trace amounts of the two antibiotics. While complete removal of CEF was always achieved, ENR appeared to be more recalcitrant. Removal percentages for this latter antibiotic varied between 40 and 55% and defluorination percentages between 3 and 65% were obtained, with biodegradation being affected by the increase in its concentration. The simultaneous supplementation of ENR and CEF did not affect the biodegradation of these antibiotics. Contrarily to what was found for ENR, abiotic mechanisms had a significant role in the removal of CEF, which may be one of the reasons why this antibiotic has a faster dissipation in the environment. Metagenomics analysis allowed to identify the major bacterial taxonomic groups associated with the biodegradation of ENR and CEF, which has not been reported before. Microbial dynamics associated to the acclimation with the target antibiotics revealed a shift in the structure of the microbial communities, with a predominant selection of microorganisms belonging to the phyla Proteobacteria (e.g., Achromobacter, Variovorax and Stenotrophomonas genera) and Bacteroidetes (e.g., Dysgonomonas, Flavobacterium and Chryseobacterium genera). Overall, this work demonstrated that microorganisms are capable of adapting and responding to the presence of different emergent pollutants, like the antibiotics used in this study, though concentration is a key factor in the biodegradation process. The capacity of the tested microbial communities to degrade ENR and CEF suggests that environmental sites contaminated with mixtures of these antibiotics are likely to be recovered through bioremediation processes. However, when evaluating the environmental fate of these antibiotics it should be taken into account key factors such as environmental concentration of the antibiotics, temperature, precipitation, UV exposure, etc. To our knowledge, this is the first study reporting biodegradation of mixtures of the veterinary antibiotics ENR and CEF and metagenomics profiles associated with the biodegradation of these two compounds. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.12.141. Acknowledgements M.F. Carvalho wishes to acknowledge Investigator FCT program supported by Fundação para a Ciência e a Tecnologia (FCT) (IF/00791/ 2013), Fundo Social Europeu and Programa Operacional Potencial Humano. W. Gao and Z. Jia acknowledge the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB15040000). This work was implemented in the framework of the structured program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-010145-FEDER-000035), namely within the research line ECOSERVICES, supported by the Northern Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). References Almeida, C.M.R., Santos, F., Ferreira, A.C.F., et al., 2017. Constructed wetlands for the removal of metals from livestock wastewater – can the presence of veterinary antibiotics affect removals? Ecotoxicol. Environ. Saf. http://dx.doi.org/10.1016/j.ecoenv. 2016.11.021.

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Please cite this article as: Alexandrino, D.A.M., et al., Biodegradation of the veterinary antibiotics enrofloxacin and ceftiofur and associated microbial community dynamics, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.141