Chemosphere 152 (2016) 301e308

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Fungal treatment for the removal of antibiotics and antibiotic

resistance genes in veterinary hospital wastewater
D. Lucas a, M. Badia-Fabregat b, T. Vicent b, G. Caminal c, S. Rodríguez-Mozaz a, *,

zar a, D. Barcelo
J.L. Balca
a, d
a
Catalan Institute for Water Research (ICRA), H2O Building, Scientific and Technological Park of the University of Girona, 17003, Girona, Spain
b
Departamentd’Enginyeria Química, Universitat Auto noma de Barcelona (UAB), 08193, Bellaterra, Spain
c
Institut de Química Avançada de Catalunya (IQAC) CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain
d
Water and Soil Quality Research Group, Department of Environmental Chemistry (IDAEA-CSIC), Jordi Girona 18-26, 08034, Barcelona, Spain

h i g h l i g h t s

Veterinary hospital effluent was treated in a fungal bioreactor.

Fungal bioreactor and control bioreactor were set up.

Comparisons with conventional treatments were established.

The best results were obtained with the fungal treatment.

a r t i c l e i n f o a b s t r a c t

Article history: The emergence and spread of antibiotic resistance represents one of the most important public health
Received 2 September 2015 concerns and has been linked to the widespread use of antibiotics in veterinary and human medicine.
Received in revised form The overall elimination of antibiotics in conventional wastewater treatment plants is quite low; there-
26 January 2016
fore, residual amounts of these compounds are continuously discharged to receiving surface waters,
Accepted 27 February 2016
Available online 15 March 2016
which may promote the emergence of antibiotic resistance. In this study, the ability of a fungal treatment
as an alternative wastewater treatment for the elimination of forty-seven antibiotics belonging to seven
Handling Edior: Chang-Ping Yu different groups (b-lactams, fluoroquinolones, macrolides, metronidazoles, sulfonamides, tetracyclines,
and trimethoprim) was evaluated. 77% of antibiotics were removed after the fungal treatment, which is
Keywords: higher than removal obtained in conventional treatment plants. Moreover, the effect of fungal treatment
Antibiotics on the removal of some antibiotic resistance genes (ARGs) was evaluated. The fungal treatment was also
Antibiotic resistance genes efficient in removing ARGs, such as ermB (resistance to macrolides), tetW (resistance to tetracyclines),
Degradation blaTEM (resistance to b-lactams), sulI (resistance to sulfonamides) and qnrS (reduced susceptibility to
Fungal treatment
fluoroquinolones). However, it was not possible to establish a clear link between concentrations of an-
Wastewater
tibiotics and corresponding ARGs in wastewater, which leads to the conclusion that there are other
Veterinary hospital
factors that should be taken into consideration besides the antibiotic concentrations that reach aquatic
ecosystems in order to explain the emergence and spread of antibiotic resistance.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction infectious diseases in human and veterinary medicine, but also as

Antimicrobial agents have been used in large quantities for
several decades since sulfonamides were introduced in the 1930s.
These compounds have been widely used not only to treat
* Corresponding author.
E-mail address: srodriguez@icra.cat (S. Rodríguez-Mozaz).
growth promoters in animal production (Sapkota et al., 2008;
Kümmerer, 2009; Zhang et al., 2009). Antibiotics may therefore
be found in different environmental compartments due to their
extensive use and the continuous drainage of surface runoff and
release from wastewater treatment plants (WWTPs) (Huerta et al.,
2013).
It is well known that antibiotics pose a significant risk to envi-
ronmental and human health, even at low concentrations

http://dx.doi.org/10.1016/j.chemosphere.2016.02.113
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
302 D. Lucas et al. / Chemosphere 152 (2016) 301e308
(Kümmerer, 2009). In addition, the overuse and misuse of antibi-
otics has led to the emergence of antibiotic-resistant bacteria,
compromising the effectiveness of antimicrobial therapy because
the infectious organisms are becoming resistant to commonly
prescribed antibiotics (Pruneau et al., 2011; Marti et al., 2014). In
fact, the emergence and spread of resistant bacteria have been
classified by the World Health Organization (WHO) as one of the
major threats to public health in the 21st century, and without
urgent actions we are heading to a post-antibiotic era, in which
common infections and minor injuries could eventually cause
death (World Health Organization, 2014).
According to recent studies, WWTPs are considered important
hotspots for the spread of antibiotic resistance (Baquero et al.,
2008; Martinez, 2009; Manaia et al., 2012), because biological
treatments, where environmental bacteria are continuously mixed
with resistant bacteria and antibiotics from anthropogenic sources,
offer an environment potentially suitable for the emergence and
spread of antibiotic resistance (Da Silva et al., 2006; Davies et al.,
2006; Auerbach et al., 2007; García-Gala
n et al., 2011). Although
the levels of antimicrobials found in WWTPs are often below
minimum inhibitory concentration, these levels may exert a se-
lective pressure on microbial populations. Several studies also
suggested that conventional technologies used in WWTPs are not
effective enough to degrade some micropollutants such as antibi-
otic compounds (Verlicchi et al., 2012a). As a consequence, some
new treatment technologies have been developed in order to
achieve higher removal efficiency of contaminants. Among them,
the fungal treatment of wastewaters can be highlighted as a
promising technology because of the unspecific enzymatic system
of ligninolytic fungi, which is able to degrade a wide range of xe-
nobiotics, including many antibiotics, present in wastewater
(Rodríguez-Rodríguez et al., 2012b; Cruz-Morato
et al., 2014; Gros
et al., 2014; Badia-Fabregat et al., 2015a, 2015b) and sludge
(Marco-Urrea et al., 2009; Rodríguez-Rodríguez et al., 2012a; Gros
et al., 2014; Llorens-Blanch et al., 2015). Data regarding specific
mechanisms, ecotoxicity and community analysis of the studied
bioreactors can be found in some of these articles (Rodríguez-
Rodríguez et al. (2012b) and Badia-Fabregat et al. (2015b)). Anti-
biotic resistance genes (ARGs) and antibiotics compounds are pol-
lutants that have different modes of action and are subject to
different fate processes in the environment (Martinez, 2009). They
are also likely to respond differently to treatment processes
designed to remove them from liquid and solid wastes (Pei et al.,
2007). Although the efficiency of ARGs removal by sewage treat-
ment procedures is such an important issue, very few studies have
addressed this topic until the last decade (Auerbach et al., 2007;
Zhang et al., 2009; Munir et al., 2011; Gao et al., 2012) and only
few of them have focused on both antibiotics and ARGs (Gao et al.,
2012; Huerta et al., 2013; Rodriguez-Mozaz et al., 2015; Xu et al.,
2015). However, none of the studies available in literature so far
have studied their fate in non-conventional biological treatments,
such as fungal treatment.
On the other hand, hospital effluents have been reported to
present a high load of antibiotics, among other pharmaceutical
compounds, and thus discussion on the suitability of some source
treatment has arose among the scientific community (Pauwels and
Verstraete, 2006; Verlicchi et al., 2010; Santos et al., 2013;
Rodriguez-Mozaz et al., 2015). The aim of this study was there-
fore to evaluate a fungal treatment of veterinary hospital waste-
water with regards to the presence of antibiotics and ARGs.
Veterinary hospital effluent was selected because it has similar
concentrations of antibiotics as human hospital effluents. A broad
range of antibiotics covering different families were selected and
monitored along the study. Culture-independent approaches were
also used to determine the prevalence of selected ARGs encoding
resistance to the main antibiotic families, such as blaTEM and blaSHV
(resistance to b-lactams), qnrS (reduced susceptibility to fluo-
roquinolones), ermB (resistance to macrolides), sulI (resistance to
sulfonamides) and tetW (resistance to tetracyclines) genes.
2. Material and methods
2.1. Fungus, wastewater and chemicals
Trametes versicolor ATCC 42530 was obtained from the American
Type Culture Collection (Manassas, VA) and maintained by sub-
culturing on malt extract agar plates at 25  C. T. versicolor was
grown in form of pellets according to a previously described
method (Bla
nquez et al., 2004).
Wastewater samples from the veterinary hospital effluent were
collected from a veterinary hospital located at the Universitat
Autonoma de Barcelona campus (Bellatera, Barcelona, Spain) on the
same day that the bioreactors were set up.
All the antibiotics and the corresponding isotopically labelled
standards were of high purity grade (>90%) and they were pur-
chased from SigmaeAldrich (Steinheim, Germany), Toronto
Research Chemicals TRC (Ontario, Canada) and CDN isotopes
(Quebec, Canada). The individual standard solutions, as well as
isotopically labelled standard solutions, were prepared according to
Gros et al. (2013). The solvents, HPLC grade methanol, acetonitrile,
water (Lichrosolv) and formic acid (98%), were provided by Merck
(Darmstadt, Germany). Glucose, ammonium tartrate dibasic, malt
extract and other chemicals were purchased from SigmaeAldrich
(Barcelona, Spain).
2.2. Fungal bioreactors and operating conditions
Two air-pulsed fluidized bed glass bioreactors (1.5 L) were set up
in parallel to treat veterinary hospital wastewater: one inoculated
with T. Versicolor in form of pellets and the other one, non-
inoculated, was used as a control with no biomass except the
wastewater-associated bacterial communities. A sterile reactor
could not be set up to measure the abiotic degradation due to the
inability to sterilize the water (heat, enzyme treatment, filtration,
etc.) without affecting the stability of antibiotics. Pellets of
T. versicolor were added at 2.0 g dry cell weight (DCW) L1. Tem-
perature was set up at 25  C and pH was controlled to be constant at
4.5 ± 0.5 by HCl 1 M or NaOH1 M addition. Bioreactors were
operated in fed-batch mode for nutrients: glucose and ammonia
tartrate were added at 277 mg g dry cell weight (DCW)1 d1 and
0.619 mg g DCW1 d1 respectively in pulses of 0.6 min h1 from a
concentrated stock. Addition rate was adjusted to avoid glucose
accumulation in the media. Glucose concentration and laccase ac-
tivity were monitored to assure the good performance of the
bioreactor, as previously described. Liquid samples of approxi-
mately 50 mL were taken at the beginning and at the end of the
experiment (after 15 days) by triplicate for the analytical pro-
cedures. The samples were kept in the dark to avoid the photo-
degradation of some of the antibiotics.
2.3. Quantification of antibiotics
Water samples were analyzed for the determination of 47 an-
tibiotics following the protocol previously described (Gros et al.,
2013). Briefly, successive filtration of water samples was done
through 2.7, 1.0 and 0.45 mm pore-size membranes (Millipore;
Billerica, MA, USA) to remove big particles that could cause prob-
lems in the analysis. After filtration, water samples of 25 mL each
were pH-adjusted to 3 with HCl (1.0 M) and EDTA (3%, v/v) and
loaded into Solid Phase Extraction (SPE)-HLB cartridges (60 mg,
 D. Lucas et al. / Chemosphere 152 (2016) 301e308
3 mL) (Waters Corp.; Mildford, MA, USA) for analytes pre-
concentration. According to the method previously mentioned,
cartridges were eluted passing 6 ml of pure methanol at a flow rate
of 2 ml min1 through the cartridges. The extracts were then
evaporated under nitrogen stream using a Reacti-Therm 18824
system (Thermo Scientific) and reconstituted with 1 mL of
methanol-water (10:90 v/v). Lastly, 10 mL of standard of internal
standard mix at 10 ng mL1 were added in the extracts for internal
standard calibration and to compensate, if it was necessary, a
possible matrix effect. Chromatographic separation of the extracted
samples was carried out with a Ultra-Performance liquid chroma-
tography system (Waters Corp. Mildford, MA, USA) equipped with a
binary solvent system (Mildford, MA, USA) and a sample manager,
using an Acquity HSS T3 column (50 mm  2.1 mm i.d. 1.8 mm
particle size; Waters Corp. Mildford, MA, USA). The UPLC instru-
ment was coupled to 5500 QqLit, triple quadrupoleelinear ion trap
mass spectrometer (5500 QTRAP, Applied Biosystems, Foster City,
CA, USA) with a Turbo V ion spray source. Analysis was performed
in positive ionization mode in a multiple reaction monitoring
(MRM) mode and the data were acquired and processed using
Analyst 2.1 software. Analysis was performed in positive ionization
mode in a multiple reaction monitoring (MRM) mode. For an ac-
curate quantification, extraction recoveries were determined in
triplicate for each sample and applied to the values obtained in the
extracts, for data correction. Those compounds, whose recovery
values did not range between 50 and 150%, were discarded.
Quantification limits (LOQs) of each compound were estimated
between 1 and 14 ng L1 (Table S3).
2.4. DNA extraction
Samples were filtered under sterile conditions through low
protein-binding 0.22-mm-pore-size membranes (Millipore). The
collected bacterial cells were then resuspended in lysis buffer (1.2%
Triton X-100, 1 M Tris-Cl, 0.5 M Na2EDTA), followed by enzymatic
digestion with lysozyme and proteinase K. Genomic DNA was
extracted using the DNeasy Blood & Tissue Kit (Qiagen; Valencia,
CA, USA), according to the manufacturer's instructions. All DNA
samples were stored at 20  C until analysis.
2.5. Quantification of ARGs
Real-time PCR (qPCR) assays were used to quantify the copy
number of selected ARGs, such as blaTEM, blaSHV, ermB, qnrS, sulI and
tetW, according to the method described by (Marti et al., 2013).
Copy number of the 16S rRNA gene was also quantified for
normalization of the data. All qPCR assays were performed using
the Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies;
Santa Clara, CA, USA), with the exception for the blaTEM gene, which
was amplified using the SYBR Green Master Mix (Applied Bio-
systems) due to nonspecific amplification. All qPCR assays were
conducted on a MX3005P system (Agilent Technologies). Each gene
was amplified using specific primer sets (Table S2) and the PCR
conditions included an initial denaturation at 95  C for 3 min, fol-
lowed by 40 cycles at 95  C for 15 s and at the annealing temper-
ature given in Table S2 for 20 s. In the case of the 16S rRNA gene,
amplification conditions were 35 cycles at 95  C for 15 s, followed
by an annealing temperature at 60  C for 1 min. A dissociation curve
was then constructed by increasing the temperature from 65 to
95  C in order to confirm the specificity of the amplified products.
Standard curves were generated by cloning the amplicon from
positive controls into the pCR2.1-TOPO vector (Invitrogen, Carlsbad,
CA, USA), and the corresponding copy number was calculated as
previously described (Rodriguez-Mozaz et al., 2015). The copy
number of each ARG was also normalized to the 16S rRNA gene
303
copy number in order to obtain relative quantification.
2.6. Statistical analysis
Mean values were compared using Student's t-test, in which
p < 0.05 was considered significant (IBM SPSS Statistics 21.0 soft-
ware; IBM, Chicago, IL, USA).
3. Results and discussion
3.1. Quantification of antibiotics
In the chemical analysis 32 out of 47 antibiotics analyzed were
detected in water samples collected from veterinary hospital used
to feed both bioreactors (Table1 and Table S3). Among them,
quinolones were detected at the highest concentration
15,701 ng L1 as sum of the 10 compounds detected from this
group, being fluoroquinolones the most concentrated ones. High
concentrations of fluoroquinolones are also found in sewage water
of human hospitals (Duong et al., 2008; Kovalova et al., 2012;
Verlicchi et al., 2012b). Such high values can be related to their
high consumption, as these compounds are frequently used in
veterinary hospitals (Riddle et al., 2000). After quinolones, the most
abundant group of antibiotics is b-lactams with an initial total
concentration of 10,253 ng L1, followed by tetracyclines, metro-
nidazoles, macrolides, trimethoprim and sulfonamides, whose
values were 4807, 4774, 309, 52 and 31 ng L1, respectively.
Despite several antibiotic compounds belong to the same family,
each of them behave differently under the same treatment pro-
cesses (Verlicchi et al., 2012b; Gros et al., 2013; Guerra et al., 2014)
and it is thus difficult to highlight a common degradation trend.
Positive removal in both bioreactors could be observed for 17 out of
32 compounds detected in the wastewater. In the case of cipro-
floxacin, enrofloxacin, marbofloxacin and ampicillin, the removal
rate achieved is significantly higher (p < 0.05) with the fungal
treatment compared with the control treatment, whereas in the
case of enoxacin, pipemidic acid, doxycycline, cefazolin, cefalexin,
chlorotetracycline, tylosin, spyramicin, ampicillin B and metroni-
dazole, no difference (p > 0.05) in degradation efficiency was
observed. The small concentrations found in hospital effluent for
tylosin, spyramicin, and chlorotetracycline prevent us to detect
significant removal differences between the two reactors.
Negative removal values were observed for clarithromycin,
erythromycin, azythromycin, danofloxacin, tilmicosin, nalixidic
acid, cinofloxacin, norfloxacin, sulfapyridyne, penicillin V and
metronidazole OH in both treatments (fungal and control) i.e., the
concentrations measured after the treatments were higher than
those found in raw water. Negative removals can be attributed to
some particular processes that take place during wastewater
treatment; many drugs e.g. clarithromycin, tetracycline and oflox-
acin are excreted conjugated with other chemical compounds
(Carballa et al., 2004; Go € bel et al., 2007; Kumar et al., 2012;
Verlicchi et al., 2012b; Jelic et al., 2015) but can be further decon-
jugated by some enzymes present in wastewater bioreactor
reverting them to their original form (Celiz et al., 2009). This effect
was also detected in other fungal bioreactors (Badia-Fabregat et al.,
2015a). Particularly remarkable is the case of sulfapyridine, usually
supplied in a conjugated form, named sulfasalazine, which is
composed of sulfapyridine conjugated to 5-aminosalicylate (Boone
et al., 1990), and that it is usually for veterinary use (Yamada et al.,
2006). That would explain why sulfapyridine was not detected in
raw wastewater but in the treated water (likely after deconjugation
processes). Another possible explanation of these negative re-
movals is related to the excretion pathway: some compounds such
as erythromycin, azythromycin, ofloxacin and trimethoprim are
304 D. Lucas et al. / Chemosphere 152 (2016) 301e308

Table 1
Concentration and removal values of the antibiotics analyzed, grouped by families.

Antibiotics Concentration±SD (ngL1) Removal (%)

t ¼ 0d With fungi t ¼ 15d Control t ¼ 15d With fungi t ¼ 15d Control t ¼ 15d

Quinolones 15,701 ± 77 1980 ± 10 9838 ± 8 87% 37%

b-Lactams 10,253 ± 7 416 ± 2 835 ± 8 96% 92%
Tetracyclines 4807 ± 24 3409 ± 11 3574 ± 0 29% 26%
Metronidazoles 4774 ± 28 4455 ± 5 5757 ± 5 7% 21%
Macrolides 309 ± 2 409 ± 1 836 ± 7 32% 171%
Trimethoprim 52 ± 11 48 ± 3 67 ± 4 8% 29%
Sulfonamides 31 ± 0 905 ± 2 908 ± 2 2846% 2856%
Total 35,927 ± 85 7119 ± 16 15,992 ± 14 77% 48%

mainly excreted with bile and faeces, so they are partly attached to

sz et al., 2009; Verlicchi
particulate matter (Lindberg et al., 2005; Plo
et al., 2012b). The load entering the bioreactors is therefore
underestimated, since it is calculated considering only the dis-
solved fraction, obviating the sorption of the antibiotics to the
suspended solids (Go €bel et al., 2007).
In the case of ofloxacin, oxytetracycline, tetracycline and
trimethoprim, negative removals were only observed in the control
treatment but not in fungal treatment, which might be attributed to
the prevalence of biodegradation processes in general, higher in
fungal than in control bioreactor in contrast to deconjugation and
desorption processes happening in both bioreactors.
Taking into account all the antibiotics measured, the removal
rates obtained with fungal treatment were in general better than
those achieved in the control bioreactor (Table 1). Overall 77% of
antibiotics were removed by fungi after 15 days, whereas only 49%
were eliminated in the uninoculated bioreactor after same period
of time. Remarkable differences were identified in the case of
quinolones: 87% removal at the inoculated and 37% at the non-
inoculated bioreactor. Macrolides also showed a big disparity be-
tween both treatments, 32% in the fungal bioreactor
against 170% at the uninoculated bioreactor. For the rest of anti-
biotic families, removal rates were either slightly better with fungal
treatment or no considerable difference was found.
The efficiency of the fungal treatment was compared with data
from conventional WWTPs for 23 antibiotics, out of the 32 found in
veterinary effluent, based on the available literature (Table S4).
Among the 23 compounds referred, 13 exhibited lower removal
rates with conventional WWTPs than with the fungal treatment
tested in our study. This is important in the case of some recalci-
trant compounds such as enrofloxacin, marbofloxacin, oxytetracy-
cline, ampicillin and cefalexin. The removal rates obtained for these
compounds in conventional WWTPs do not exceed 40% (39, 146,
9, 7 and 38%, respectively). Meanwhile with the fungal treatment
the values obtained are notably better: 76, 87, 57, 100 and 98%,
respectively (Table S4).
3.2. Quantification of ARGs
Six ARGs, blaTEM, blaSHV, ermB, qnrS, sulI and tetW, and the 16S
rRNA gene were quantified using qPCR assays in water samples.
High R2 (average 0.991) and efficiency values (from 85 to 108%)
were obtained from the standard curves showing the linearity and
the sensitivity of each qPCR assay (Table S5). The 16S rRNA gene
was also analyzed to quantify the total bacterial load and to
normalize the abundance of ARGs in the collected samples.
Four trends were observed regarding the presence of ARGs
before and after fungal treatment (Fig. 1). The first one corre-
sponds to complete disappearance of the ermB and tetW genes,
both after fungal treatment and in the non-inoculated control
bioreactor. Previous studies have also demonstrated a complete
removal of these resistance genes in conventional WWTPs (Munir
et al., 2011; Gao et al., 2012; Rodriguez-Mozaz et al., 2015). A
second trend is represented by the blaTEM gene, in which the
fungal treatment resulted in a marked decrease in the copy
number of this gene. Likewise, similar observations have been
found for this gene after conventional wastewater treatment
(Lachmayr et al., 2009; Rodriguez-Mozaz et al., 2015). In contrast,
no significant difference (p ¼ 0.256) was found between samples
before and after experimental period in the non-inoculated con-
trol bioreactor. The third pattern corresponds to the blaSHV and sulI
genes, whose copy numbers increased in both bioreactors; how-
ever, the increase was significantly lower (p < 0.05) in the fungal
bioreactor than in the control bioreactor. In fact, an approximately
thousand-fold and ten-fold increase, of these blaSHV and sulI genes,
was observed in the fungal bioreactor, whereas a hundred-fold
reduction was observed in conventional WWTPs for the sul I
gene (Yuan et al., 2014; Rodriguez-Mozaz et al., 2015). Regarding
blaSHV gene, a reduction has been found in some WWTPs, whereas
no reduction has been detected in another one (Laht et al., 2014).
Finally, the qnrS gene presents an increase in the copy number in
both treatments; however, no statistically significant difference
was found between both bioreactors (p ¼ 0.56). Opposite trends
were obtained in conventional WWTPs (Rodriguez-Mozaz et al.,
2015) in which the qnrS gene decreases its copy number almost
completely.
The increase of the sul I, blaSHV and qnrS genes observed in our
studies can be related to the increase of total bacteria (as measured
by 16S rRNA gene copy number). In fact, a ten-fold increase in the
copy numbers of 16S rRNA genes was observed in both bioreactors,
whereas data from conventional WWTPs showed ten- (Lachmayr
et al., 2009), hundred- (Rodriguez-Mozaz et al., 2015), and even
thousand-fold (Gao et al., 2012) reductions for these genes. The
increase of bacterial population in our experiments can be attrib-
uted to the special conditions in which the bioreactors were set up
to facilitate the growth of the fungus: nutrients addition, stable pH
and temperature. It should also be noted that the increase of the
16S rRNA gene was slightly higher in the inoculated bioreactor,
possibly due to the presence of the fungi, which produce extra-
cellular degradative enzymes, e.g. laccase and peroxidases (Mougin
et al., 2002; Cruz-Morato
et al., 2013). These enzymes break com-
plex compounds increasing the nutrients bioavailability for all
microbial community members.
Therefore, according to the normalized data (Fig. 2), the fungal
treatment showed better relative removal rates for ARGs than those
obtained from a conventional WWTP (Table 2). In fact, the fungal
treatment resulted in a good removal of the sulI and blaTEM genes,
reaching removal rates of 56 and 100%, respectively, as compared
with data obtained from a conventional WWTP, in which the
removal rates were negative, 156 and 58%, respectively
(Lachmayr et al., 2009; Gao et al., 2012; Rodriguez-Mozaz et al.,
2015). The qnrS gene showed a large difference between the
 D. Lucas et al. / Chemosphere 152 (2016) 301e308
Fig. 1. Absolute concentration of ARGs in the different samples analyzed. Within the box plot chart the crosspieces of each box plot represent (from top to the bottom) maximum,
upper-quartile, median (white bar), lower-quartile and minimum values.
treatments, but with negative removal values in both of
them; 163% for the fungal treatment and 302% for the conven-
tional WWTPs (Rodriguez-Mozaz et al., 2015). The difference be-
tween treatments is not so remarkable with the ermB and tetW
genes; however, better removal rates have been obtained with the
fungal treatment (100% for both genes) than at conventional
WWTPs (82 and 87%, respectively) (Gao et al., 2012; Rodriguez-
Mozaz et al., 2015). Finally, the removal efficiency of the blaSHV
gene also showed a large difference between the fungal treatment
(843%) and the values obtained from conventional WWTPs (33%)
(Laht et al., 2014). Despite these differences, the absolute copy
numbers were 1.4  105 in the fungal treatment and 8  106 in the
conventional WWTP at the end of each treatment showing that an
important point for removal evaluation is also always the initial
concentration.
3.3. Relationship between antibiotics and ARGs
In view of the differences in behavior found for the ARGs and in
order to reach a better understanding of the processes involved in
the spread of antibiotic resistance, a possible correlation between
antibiotics and ARGs has been studied. Unfortunately, the number
of samples did not allow carrying out a statistical correlation
analysis. Nevertheless with experimental data and those obtained
305
from the literature, some ideas could be highlighted regarding each
ARG (Fig. 3).
The first association was found between the ermB gene and
macrolides. Whilst the gene disappeared completely in both bio-
reactors, macrolides experienced a slight increase in their con-
centration. This observation, contrary to the classical knowledge
(Davies, 1994; Allen et al., 2010), is in agreement with the findings
of a recent study (Rodriguez-Mozaz et al., 2015), where the con-
centration of the ermB gene decreased by 3 orders of magnitude in
the presence of even higher concentrations of macrolides.
The tetW gene disappeared totally in both bioreactors, even
though tetracycline antibiotics were hardly removed along the
treatment (29 and 26% removal in the fungal and the control bio-
reactors, respectively). The concentration of this gene has also been
reported to decrease by three or four orders of magnitude in
presence of small amounts of tetracycline antibiotics, although
higher than those detected in the bioreactors in this work (Wu
et al., 2010; Gao et al., 2012; Xu et al., 2015).
The concentration of b-lactam antibiotics in raw wastewater
was quite high (c.a. 10 mg L1) although removal in both bioreactors
was very efficient, reaching values close to zero. Nevertheless,
levels of blaTEM and blaSHV along the treatment were quite different.
In the fungal bioreactor, the blaTEM gene also disappeared (100%
removal), in agreement with previous studies (Rodriguez-Mozaz
306 D. Lucas et al. / Chemosphere 152 (2016) 301e308

Fig. 2. Relative concentration of ARGs in the different samples analyzed. Within the box plot chart the crosspieces of each box plot represent (from top to the bottom) maximum,
upper-quartile, median (white bar), lower-quartile and minimum values.

Table 2 decreased their concentrations. These positive correlations be-

Comparison of relative removal rates of the genes analyzed between this study and tween the gene and antibiotics are in line with the classical
previous reports.
knowledge about the emergence of antibiotic resistance (Davies,
Treatments ermB qnrS sul I tetW blaTEM blaSHV 1994; Allen et al., 2010).
Fungal treatment 100% 163% 56% 100% 100% 843% The relationship between the qnrS gene and quinolones showed
Conventional WWTPa 82% 302% 58% 87% 156% 33% a similar trend to that found between the blaSHV gene and b-lactams.
a
Mean value of data obtained from Lachmayr et al., 2009; Gao et al., 2012 and An increase of the qnrS genes was observed, whereas the antibiotic
Rodriguez-Mozaz et al., 2015. decreased. Quinolones are the most abundant group in wastewater
and therefore, despite their depletion, they may exert enough se-
lective pressure to increase the gene concentration. Some studies
et al., 2015), whereas ARG concentration in control bioreactor did have also suggested that qnr genes may have other functions (e.g.
not undergo noteworthy change after treatment. In contrast, the regulation of cellular DNA-binding proteins) in addition to the
blaSHV gene increased in both bioreactors almost to the same antibiotic resistance that contribute to its spread (Wang et al., 2004).
extent, in agreement with the assumption that ARGs increase is
favored by the presence of selective agents, such as antibiotics
4. Conclusions
(Davies, 1994; Allen et al., 2010). The hypothesis here is that despite
the decrease in the concentration of b-lactams in both bioreactors,
In this study, antibiotics and ARGs from the effluent of a veter-
the concentration was high enough to exert a selective pressure;
inary hospital were measured in order to analyze the efficiency of
however further studies are required to understand the relation-
an alternative wastewater treatment. Based on removal rates of
ship between the evolution of the blaTEM and blaSHV genes and the
both antibiotics and ARGs, fungal treatment emerges as an inter-
concentration of b-lactam antibiotics, including the exposure to
esting technology; however, an optimization or combination with
sub-therapeutic concentrations.
other methods is needed in order to reduce the amount of associ-
The concentration of sulfonamides and the sul I gene increased
ated microbiota. This treatment offers good results in terms of
in both bioreactors, whereas in another study in a urban WWTP
elimination of certain compounds such as ciprofloxacin, enro-
(Rodriguez-Mozaz et al., 2015) both antibiotics and the gene
floxacin, marbofloxacin and ampicillin, which are recalcitrant in
 D. Lucas et al. / Chemosphere 152 (2016) 301e308
Fig. 3. Absolute concentration of a) ARGs and b) antibiotics grouped into families.
conventional WWTPs.
It has also been observed that the presence of antibiotic com-
pounds is not the only factor influencing the fate of antibiotic
resistance. Therefore, other factors should also be taken into ac-
count such as the operational parameters of bioreactors, the
wastewater-associated bacterial communities and their interaction
with fungi, which may contribute to the spread of resistance genes
associated with certain families of microorganisms.
Acknowledgments
The authors wish to acknowledge the UAB veterinary hospital staff
for their kindness permission and help for the samplings. This work
has been funded by the Spanish Ministry of Economy and Competi-
tiveness (projects CTM2013-48545-C2-2-R and JPIW2013-089-C02-
02), co-financed by the European Union through the European
Regional Development Fund (ERDF) and also supported by the Gen-
eralitat de Catalunya (Consolidated Research Groups 2014-SGR-476
and 2014-SGR-291). D. Lucas and M. Badia-Fabregat acknowledge the
predoctoral grants from the Spanish Ministry of Education, Culture
and Sports (AP-2010-4926) and from UAB, respectively. J.L Balcazar
and Sara Rodriguez-Mozaz acknowledge the Ramo
n y Cajal program
(RYC-2011-08154 and RYC-2014-16707, respectively).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2016.02.113.
307
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