Science of the Total Environment 605–606 (2017) 1047–1054

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Sewers as potential reservoirs of antibiotic resistance

Olga Auguet a, Maite Pijuan a, Carles M. Borrego a,b, Sara Rodriguez-Mozaz a, Xavier Triadó-Margarit b,c,
Saulo Varela Della Giustina a, Oriol Gutierrez a,⁎
a
Catalan Institute for Water Research (ICRA), Scientific and Technologic Park of the University of Girona, Emili Grahit 101, 17003 Girona, Spain
b
Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain
c
Integrative Freshwater Ecology Group, Centre d'Estudis Avançats de Blanes, CEAB-CSIC, Accés Cala Sant Francesc, 14, 17300, Blanes, Girona, Spain

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

• Occurrence of antibiotic compounds in

sewer biofilm and wastewater has
been shown.
• ARGs linked to the main antibiotic fam-
ilies were detected in sewage and
sewer biofilm.
• Sewers are likely hotspots for antibiotic
resistance accumulation and spread.

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

Article history: Wastewater transport along sewers favors the colonization of inner pipe surfaces by wastewater-derived microor-
Received 6 June 2017 ganisms that grow forming biofilms. These biofilms are composed of rich and diverse microbial communities that
Received in revised form 19 June 2017 are continuously exposed to antibiotic residues and antibiotic resistant bacteria (ARB) from urban wastewater.
Accepted 19 June 2017
Sewer biofilms thus appear as an optimal habitat for the dispersal and accumulation of antibiotic resistance genes
Available online xxxx
(ARGs). In this study, the concentration of antibiotics, integron (intI1) and antibiotic resistance genes (qnrS, sul1,
Editor: D. Barcelo sul2, blaTEM, blaKPC, ermB, tetM and tetW), and potential bacterial pathogens were analyzed in wastewater and biofilm
samples collected at the inlet and outlet sections of a pressurized sewer pipe. The most abundant ARGs detected in
Keywords: both wastewater and biofilm samples were sul1 and sul2 with roughly 1 resistance gene for each 10 copies of 16s
Sewers RNA gene. Significant differences in the relative abundance of gene intI1 and genes conferring resistance to
Antibiotics fluoroquinolones (qnrS), sulfonamides (sul1 and sul2) and betalactams (blaTEM) were only measured between
Antibiotic resistance genes inlet and outlet biofilm samples. Composition of bacterial communities also showed spatial differences in biofilms
Wastewater and a higher prevalence of Operational Taxonomic Units (OTUs) with high sequence identity (N 98%) to well-
Biofilm
known human pathogens was observed in biofilms collected at the inlet pipe section. Our study highlights the
role of sewer biofilms as source and sink of ARB and ARGs and supports the idea that community composition rather
than antibiotic concentration is the main factor driving the diversity of the sewage resistome.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. In urban systems, the main anthropogenic source of antibiotics is

E-mail address: ogutierrez@icra.cat (O. Gutierrez). human waste. Only a fraction of the used pharmaceutical products are

http://dx.doi.org/10.1016/j.scitotenv.2017.06.153
0048-9697/© 2017 Elsevier B.V. All rights reserved.
1048 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054
metabolized within the human body and a large part of the parent com-
pound and/or its metabolites (between 20 and 97%) are excreted
through urine and feces (Jelic et al., 2015). Urban wastewater thus con-
tains not only a large repertoire of antibiotic residues but also antibiotic
resistant bacteria (ARB) from human origin (Baquero et al., 2008) that
are transported along sewer systems. Sewers are an underground net-
work of physical structures-installations (pipelines, pumping stations,
manholes and channels) that convey wastewater from its source to
the discharge point, usually a wastewater treatment plant (WWTP). Al-
though sewers have traditionally been considered as passive transport
systems, they are hot spots of microbial diversity and activity. Moreover,
sewers are the first section of the urban wastewater cycle and thus the
most likely compartment where excreted antibiotic residues may stim-
ulate resistance among microbes and their further accumulation and
spread. In sewers, wastewater-derived microorganisms grow attached
to inner pipe surfaces forming biofilms (Hvitved-Jacobsen et al.,
2013). Biofilms are characterized by very diverse microbial communi-
ties, high cell densities and close contact between cells that provide op-
timal conditions for trophic interactions and horizontal gene transfer
among community members (Taylor et al., 2011). Sewer biofilms not
only alter wastewater chemistry but also contribute to relevant process-
es such as sulfide and methane emissions (Auguet et al., 2016, 2015a,
2015b; Jiang et al., 2015).
Despite the wealth of information on the effectiveness of WWTPs on
the removal of antibiotic residues, antibiotic resistant bacteria (ARBs)
and antibiotic resistance genes (ARGs) during wastewater treatment
and its consequences for human and environmental health (Baquero
et al., 2008; Pruden, 2013; Pruden et al., 2012; Rodriguez-Mozaz et al.,
2015), less data are available on the abundance of these targets within
sewers. Moreover, although sewer biofilms are constantly exposed to
antibiotic residues and challenged by wastewater-derived ARB no infor-
mation is available on which of these factors would contribute the most
to enriching the sewer resistome. Most studies generally use sewage
samples (i.e. WWTP influents) as reference to evaluate removal rates
for antibiotics (Collado et al., 2014; Jelic et al., 2011; Zorita et al.,
2009), ARB and ARGs (Bengtsson-Palme and Larsson, 2016;
Bengtsson-Palme et al., 2016; Korzeniewska et al., 2013; Rizzo et al.,
2013; Schwartz et al., 2003) during wastewater treatment. To the best
of our knowledge no studies have addressed simultaneously the occur-
rence and potential variations in the concentration of antibiotics and
ARGs along sewer pipes and their links to spatial variations on the com-
position of bacterial communities (including pathogens) inhabiting dif-
ferent sewer compartments (i.e. wastewater and biofilms).
Our main goal was to assess the abundance of ARGs along a sewer
pipe in relation to the antibiotic burden and the bacterial diversity at dif-
ferent sewer compartments (wastewater and biofilms). The study was
carried out in a full-scale sewer in the municipality of Palamós (Catalo-
nia, Spain) that collects urban wastewater. The abundance of antibiotic
Fig. 1. Location and layout of the studied sewer. Point 1 (SP1): pump station collecting wastewater; Point 2 (SP2): wastewater treatment plant inlet.
residues and genes encoding resistance to fluoroquinolones,
betalactams, sulphonamides, macrolides and tetracyclines, and a gene
encoding the Class I integron integrase involved in gene capture and
mobilization, were quantified in wastewater and biofilms at both ends
of the sewer pipe. The composition of wastewater and biofilm bacterial
communities, including the presence of potential pathogens, was also
determined to investigate their links with the changes observed in the
abundance of ARGs along the studied sewer.
2. Materials and methods
2.1. Description of the studied system
The studied collection system is situated in municipality of Palamós,
in the northeastern coastal region of Catalonia (Spain) (Fig. 1). The sys-
tem consists of a pump station (Point SP 1) that collects wastewater of
the suburb of Sant Antoni de Calonge (South-East Palamós) and pushes
it to the Palamós WWTP (Point SP2). The length of the sewer pipe is
4800 m with an internal diameter of 556 mm and a variable Hydraulic
Retention Time (HRT) of 9–15 h. The average daily flow during the sam-
pling period was 2812 ± 77 m3 sewage/day, which accounts for an es-
timated population equivalent of ≈14,000 inhabitants, assuming that a
person produces 200 L of wastewater per day. The average volume of
sewage pumped per event was 23.3 m3, with pump events lasting
around 4 min. Infiltration and exfiltration were considered negligible
as the pipe has no lateral streams and is in good condition.
2.2. Sample collection
Composite samples of wastewater were collected during a 24-hour
period using a flow-proportional sampling mode following the sam-
pling guidelines proposed by Ort et al. (2010). Two points, respectively
named SP1 for the inlet section of the pipe and SP2 for the outlet section
of the pipe, were sampled. The start of sampling at SP2 (WWTP influ-
ent) was always delayed for 9–15 h (depending on the different HRT
of the pipe in each period of the day) in order to capture the portion
of the wastewater stream measured at SP1. The sampling was carried
out over 3 consecutive days in September 2015. The weather was dry
and stable, with the environmental temperatures ranging from 15 °C
to 30 °C. Samples were collected employing 2 portable automatic refrig-
erated samplers Hach-Lange Buhler BL 2000 with 24 PE containers of
1 L. The autosamplers were programmed to collect a 140 mL sample
every 10 min to obtain a 20.1 ± 0.2 L composite daily. The frequency
of pump events and the HRT variation are shown in Fig. S1. The
uncertainty due to sampling was estimated by considering the
population served by the sewer and their pharmaceutical consumption
pattern, and the applied sampling procedure as proposed in the
 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054
literature (Ort et al., 2010). Uncertainty associated with the sampling
was always below 5% (see SI for details and Table S1).
For molecular analysis of biofilm samples, triplicate samples were
taken from SP1 and SP2 points once wastewater sampling was complet-
ed to prevent any disruption of the system. Normal functioning of the
system was temporarily stopped to collect biofilm samples. In SP1, bio-
film was removed from a submerged air scour valve which was perma-
nently in contact with wastewater. In SP2, biofilm was removed from
the inner walls of the pipe 1.5 m before wastewater discharge into the
treatment plant. Collected samples were maintained at 4 °C to avoid bi-
ological degradation. The samples were extracted and processed within
12 h.
2.3. Analysis of antibiotics
A total of 18 wastewater composite samples (triplicates of wastewa-
ter at SP1 and SP2 over 3 days) were filtered through 1 μm glass fiber fil-
ter and 0.45 μm Polyvinylidene fluoride fiber filter. Wastewater samples
were analyzed in triplicate for the determination of 53 antibiotics ac-
cording to a previous reported method (Gros et al., 2013). Detailed in-
formation about the method used is given in the SI.
The uncertainty associated with the measured antibiotic concentra-
tions was calculated from the individual uncertainties from antibiotic
chemical analysis and from sampling. The uncertainty of chemical anal-
ysis was estimated from the relative recoveries, triplicate analysis of the
samples, and other uncertainty factors (i.e. 2%, (Kovalova et al., 2012)).
The mixture of chemical standards was prepared just before the analy-
sis, so the error associated with the stability of the solution can be con-
sidered negligible.
For the analysis of antibiotic residues in biofilms, samples were gent-
ly scraped from the surface of the submerged valve (SP1) and the inner
surface of the sewer pipe (SP2) and placed directly into sterile Falcon®
tubes and transported to the laboratory in a portable icebox in the dark.
Excess water was removed by centrifugation and the pelleted biofilm
biomass was freeze-dried and kept frozen (−20 °C) until analysis. Bio-
film samples were analyzed in triplicate for the determination of 33 an-
tibiotics adapted from a previously reported method (Huerta et al.,
2016). Details of protocols for biofilm extraction, purification and anal-
ysis of antibiotics are summarized in SI. Detailed information on the
chemicals used including analytical quality parameters of the method
and the preparation of the mixture solutions is given in Table S2.
2.4. DNA extraction and quantification of antibiotic resistance genes
Composite wastewater samples of both sampling points were im-
mediately fixed with an RNA preservation solution after its collection
(5% water-saturated phenol in absolute ethanol, using a 1:10 dilution
of fixation solution) (Feike et al., 2012) and preserved at 4 °C during
the 3 days of sampling. These samples (30 mL) were centrifuged at
11,000 rpm for 10 min at 4 °C in an Eppendorf 5804R centrifuge
equipped with an F-34-6-38 rotor (Eppendorf, Hamburg, Germany)
and the resulting pellet was stored at −80 °C. Biofilm was resuspended
in 3 mL of LifeGuard™ Soil Preservation Solution (MO BIO Laboratories,
Inc., Carlsbad, CA) and immediately frozen at −80 °C until DNA extrac-
tion. Nucleic acids were extracted from collected biofilm samples using
FastDNA® SPIN kit according to manufacturer instructions.
We quantified gene copies of eight ARGs, namely: i) qnrS (reduced
susceptibility to fluoroquinolones); ii) sul1 and sul2 (resistance to sul-
fonamides); iii) blaTEM and blaKPC (resistance to β-lactams); iv) ermB
(resistance to macrolides), and tetM and tetW (resistance to tetracy-
clines). In addition, the gene encoding the integrase of Class I integrons
(intI1) was also quantified as a proxy for the potential capacity of the
bacterial community to disseminate resistance (Boucher et al., 2007;
Ma et al., 2017). The selected ARGs were quantified in the wastewater
and biofilm DNA extracts by quantitative PCR (qPCR). Information
1049
about qPCR analysis, primers used and amplification conditions used
are provided in SI and Table S3.
2.5. High-throughput sequencing and sequence processing
High-throughput multiplexed 16S rRNA gene sequencing with the
Illumina MiSeq System (2 × 250 PE) was carried out using primer pair
515f/806r (Caporaso et al., 2011) targeting the V4 region of the 16S
rRNA gene complemented with Illumina adapters and sample-specific
barcodes at the genomics core facilities of the Research Technology Sup-
port Facility Michigan State University, USA (Kozich et al., 2013). Raw
forward and reverse paired sequences were merged, quality filtering,
chimera checking, Operational Taxonomic Unit (OTU) clustering (97%
cutoff) removing singletons, identification of representative OTU se-
quences and construction of the OTU table were also carried out in
UPARSE (Edgar, 2013). The resulting OTU table was imported and ana-
lyzed into QIIME (Caporaso et al., 2010), which was used for the align-
ment and taxonomic assignment of representative OTU sequences. For
community analysis, the number of sequences in each sample was nor-
malized by randomly selecting a subset of 44,000 sequences per sample
to standardize sequencing effort across samples. QIIME was also used to
calculate α-diversity indicators (Chao1 and Shannon Index) and to cal-
culate similarity between bacterial communities (β-diversity). Signifi-
cance of the difference in community composition between sample
categories (e.g. wastewater vs. biofilm) was assessed by Permanova
(999 permutations) in QIIME. Details of the analytical pipeline are pro-
vided in the SI. Raw sequencing data of this study have been deposited
in the NCBI database under accession number PRJNA384591 (http://
www.ncbi.nlm.nih.gov/bioproject/).
2.6. Analysis of potential pathogens
The presence of potentially pathogenic bacteria in wastewater and
biofilm samples was analyzed using tBLAST against a de-novo created
database of 16S rRNA gene sequences representing 283 bacterial strains
of obligated and opportunistic pathogens as reported previously
(Triado-Margarit et al., 2016). The searched database was constructed
from the list of pathogenic species inventoried in a previous work
(Ecker et al., 2005) and further refined by means of on-line searches
of the ascribed taxa. For the current analyses, just those OTUs with
both sequence identity values and BLAST alignment coverage ≥ 98%
were considered as potential pathogens. It should be noted, however,
that even OTUs with identity values of 100% would need additional con-
firmation steps (e.g. by studying specific virulence markers) to fully cor-
roborate the pathogenic nature of the target strain. Accordingly, results
presented here should be only regarded as indicative of potentially
pathogenic bacteria and they should be interpreted with caution.
OTUs ascribed to potential pathogenic bacteria were further classified
in accordance to their host, general categories related to affectation or
pathogenic potential (obligated or opportunistic).
2.7. Statistical analysis
The relative abundance of targeted ARGs (ARG copies/16S rRNA
gene copies) were log-transformed before analysis. Transformed data
were assessed for normality in R (R Development Core Team, 2016). R
was also used to assess for significant differences between sampling
sites (upstream and downstream section of the pipe) using pairwise
Student's t-test (package stats) and correction of p-values for multiple
testing using the false discovery rate (Benjamini and Hochberg, 1995)
with a significance cutoff of 0.05. Data on community composition and
occurrence of putative pathogens were plotted in R using package
ggplot2 (Wickham, 2009). Ordination of samples according to Bray-
Curtis distance matrices on ARGs abundance was done using Principal
Coordinate Analysis (PCoA) in PRIMER 6 statistical package with the
PERMANOVA+ add-on (PRIMER-E, Plymouth Marine Laboratory, UK).
1050 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054
3. Results and discussion
3.1. Occurrence of antibiotics in wastewater and sewer biofilms
Fig. 2 shows the average concentration of the detected antibiotics in
the flowing wastewater and biofilms during the three different days of
sampling, both in at the inlet and outlet of the pipe. Only 9 out of the
total 53 antibiotics analyzed were detected in the wastewater (all re-
sults are compiled in Table S4) with fluoroquinolones (ofloxacin,
enrofloxacin, norfloxacin and ciprofloxacin) being the most prevalent
family (Fig. 2A). The highest concentrations detected corresponded to
ciprofloxacin (from 2905 to 1379 ng/L), norfloxacin (from 652 to
731 ng/L) and ofloxacin (from 421 to 649 ng/L). The concentration of
the different antibiotic residues in the wastewater from the studied
sewer were similar to those reported by other authors in sewers and
WWTP influents despite the large variability in absolute concentrations
observed in some systems (Dong et al., 2016; Gros et al., 2013; Jelic
et al., 2015; Verlicchi et al., 2012). Remarkably, some compounds such
as ciprofloxacin and amoxicillin decreased their concentration during
its passage along the pipe (Fig. 2A), agreeing with results obtained in a
similar pressure sewer system (Jelic et al., 2015). In that study, the de-
crease in the concentration of pharmaceuticals was hypothesized to
occur due to a combination of different processes such as chemical
and physical transformation, biodegradation and sorption to solid mat-
ter (Jelic et al., 2015).
No information is available about the abundance of antibiotics in
biofilms from sewer pipes. In this compartment, ofloxacin, norfloxacin
and ciprofloxacin were the most prevalent antibiotics measured within
biofilms agreeing with results from the flowing wastewater (Fig. 2B).
Several studies have pointed out that fluoroquinolones are easily
retained in sludge and particulate matter during wastewater treatment
(Jelić et al., 2012; Petrie et al., 2015). Besides, the biofilm matrix is main-
ly composed of extracellular polymeric substances that are known to
play an important role in sorption of different compounds from the
water phase (Dobor et al., 2012; Huerta et al., 2015). Contrary to results
from wastewater, antibiotic concentrations in biofilms were higher at
Fig. 2. Concentration of antibiotics (mean of triplicate measurements ± SEM) measured at
inlet and outlet sections of the sewer pipe in (A) wastewater and (B) biofilms. In the upper
graph, values below bars correspond to the concentration of the antibiotic (in ng/L) that
selects for resistance (Korzeniewska et al., 2013). n.d.: not determined.
the outlet sections of the pipe compared to those at the inlet section
(Fig. 2B, all results are compiled in Table S5). Since these differences
could not be attributed to wastewater characteristics, variations in the
overall structure of the biofilm matrix (e.g. thickness, content of extra-
cellular polysaccharides) at the outlet sections may be responsible for
the differential accumulation of antibiotic residues. According to the re-
sults, the chronic exposure to antibiotic residues from wastewater favor
the selective accumulation of some of these compounds into the biofilm
matrix as observed in river biofilms impacted by WWTP discharges
(Huerta et al., 2015; Writer et al., 2013). Further research is needed to
resolve if sewer biofilms effectively remove antibiotics from wastewa-
ter, the specificity of this removal and its dependency on biofilm
characteristics.
Further to the previous point, an important aspect regarding the
concentrations of antibiotics measured in the studied sewer is if they
are expected to select for resistance in resident bacteria (either in the
flowing wastewater or in the biofilms). Comparison of the absolute con-
centrations of antibiotics measured in wastewater to the predicted con-
centrations that select for resistance in environmental bacteria
(Bengtsson-Palme et al., 2016) showed that selection thresholds were
exceeded for quinolones, clarithromycin, amoxicillin and sulfamethoxa-
zole (Fig. 2A). Although quinolones concentration were above 0.5 μg/g
in biofilms (Fig. 2B), direct comparisons to selection thresholds should
be taken cautiously due to the different sample matrix. According to
these figures, the antibiotic concentrations measured in the studied sys-
tem (Table S5) were high enough to exert a selective pressure on
wastewater-derived microorganisms to favor the emergence and
growth of resistant phenotypes.
3.2. Quantification of antibiotic resistance genes in sewer samples
All target genes were detected in both studied compartments and
showed remarkable differences in their relative concentrations
(Table 1). The most abundant ARGs detected in both wastewater and
biofilm samples were sul1 and sul2 with roughly 1 resistance gene for
each 10 copies of 16 s RNA gene. Genes conferring resistance to sulfon-
amides have consistently been the most abundant ARGs detected in raw
sewage (Laht et al., 2014; Luprano et al., 2016; Xu et al., 2015). All other
ARGs showed a lower but still substantial presence. Genes conferring
resistance to macrolides (ermB) and tetracyclines (tetM and tetW)
were measured at similar concentrations (≈−2 log(ermB copies/16S
rRNA copies) in both studied habitats.
These values are slightly higher than those measured in raw waste-
water for ermB by Luprano et al. (2016) and for tetW in influents of dif-
ferent WWTP in China (Gao et al., 2012; Xu et al., 2015). Differences in
the concentration of genes conferring resistance to betalactams be-
tween wastewater and biofilms were also remarkable (Table 1). More-
over, the gene conferring resistance to carbapenems (blaKPC) showed
the lowest relative abundance of all studied genes in both compart-
ments. Concerning resistance to fluoroquinolones, slight differences in
the relative concentration of qnrS were measured between the flowing
wastewater and the biofilm. The lower concentrations of qnrS in
biofilms are unexpected considering that fluoroquinolones were the
most abundant antibiotics measured in this compartment (Fig. 2). In
this regard, several studies have highlighted the lack of correlation be-
tween the abundance of ARGs and the concentration of antibiotics to
which these genes confer resistance to (Ben et al., 2017;
Bengtsson-Palme et al., 2016) although in some cases the significance
of these correlations is case dependent (Rodriguez-Mozaz et al., 2015;
Xu et al., 2015).
Comparison of the relative abundance of ARGs in sewer biofilms
with data from freshwater biofilms exposed to WWTP discharges
yielded contrasting results depending on the resistance gene. The rela-
tive concentration of sul1 and blaTEM in sewer biofilms were roughly
one order of magnitude higher than that in river biofilms (Marti et al.,
2013; Proia et al., 2016). In turn, both type of biofilms showed similar
 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054 1051

Table 1
Relative concentration of ARGs (log[ARG copies/16S rRNA gene copies]) in wastewater and biofilm samples collected at the different sections of the sewer pipe. Values correspond to the
mean of biological triplicates ± standard error or the mean. The p-values of pairwise t-test between inlet and outlet samples are also shown. Significant p-values are shown in bold
typeface.

Gene Wastewater pa Biofilm pa

Inlet Outlet Inlet Outlet

intI1 −1.15 ± 0.02 −1.14 ± 0.03 0.859 −1.16 ± 0.05 −1.60 ± 0.01 0.014
qnrS −2.03 ± 0.02 −2.13 ± 0.06 0.231 −2.69 ± 0.02 −3.30 ± 0.02 4.5e−05
sul1 −0.89 ± 0.01 −0.86 ± 0.03 0.404 −0.73 ± 0.07 −1.12 ± 0.04 0.040
sul2 −1.18 ± 0.04 −1.15 ± 0.01 0.669 −1.39 ± 0.04 −1.68 ± 0.02 0.016
blaTEM −2.45 ± 0.04 −2.53 ± 0.08 0.460 −3.08 ± 0.12 −3.66 ± 0.02 0.048
blaKPC −4.57 ± 0.12 −4.64 ± 0.08 0.658 −4.80 ± 0.14 −5.21 ± 0.09 0.148
ermB −1.97 ± 0.04 −2.08 ± 0.08 0.390 −2.26 ± 0.09 −2.13 ± 0.05 0.390
tetM −2.93 ± 0.02 −2.98 ± 0.01 0.540 −2.99 ± 0.04 −3.02 ± 0.04 0.760
tetW −2.13 ± 0.03 −2.16 ± 0.06 0.750 −2.32 ± 0.05 −2.42 ± 0.01 0.230
a
p-Values are adjusted for multiple comparisons using the false discovery rate method (Benjamini and Hochberg, 1995).

figures for the relative concentration of qnrS and ermB genes. In waste-
water, the concentration of qnrS genes was also similar to those mea-
sured in hospital effluents, WWTP influents and effluents by
Rodriguez-Mozaz and co-workers (Colomer-Lluch et al., 2014;
Rodriguez-Mozaz et al., 2015). The prevalence of a given resistance
gene over the others is probably ruled by a multifactorial process, in-
cluding nutrient concentration, antibiotic and metal pollution and,
eventually, by the composition of the bacterial community
(Bengtsson-Palme et al., 2016).
Collection of samples at the inlet and the outlet sections of the sewer
allowed us to assess for potential differences in ARG abundance be-
tween both ends. Significant differences between the inlet and the out-
let sections were measured for intI1 and for 4 out of the 8 studied ARGs,
namely qnrS, sul1, sul2 and blaTEM (Table 1). Remarkably, biofilm sam-
ples collected at the outlet section consistently had lower relative con-
centrations of the target genes than those collected at the inlet
section. In turn, no statistical differences in the relative concentrations
of ARGs were measured between wastewater collected from inlet and
outlet pipe sections. These spatial differences were clearly visible
when samples were ordinated according to the relative concentration
of ARGs (Fig. S2A). Remarkably, same grouping was obtained when
samples were ordinated according to the phylogenetic composition of
bacterial communities (Fig. S2B, see next section). These results agree
with those obtained by Bengtsson-Palme and co-workers regarding
the leading effect of community composition on the abundance and di-
versity of the wastewater resistome (Bengtsson-Palme et al., 2016).
3.3. Composition of wastewater and biofilms bacterial communities
Sewer biofilms were, in turn, characterized by a large percentage of
sequences affiliated to Chloroflexi, Firmicutes and, specially,
Deltaproteobacteria and Synergistia. PERMANOVA test revealed signifi-
cant differences in community composition between wastewater and
biofilm samples (p b 0.004) although no significance was obtained
when these communities were compared according to the collection
site (PERMANOVA, p = 0.104 and p = 0.102 for wastewater and
biofilms, respectively). Despite this overall similarity, indicators of rich-
ness (Chao1) and diversity (Shannon index) of bacterial communities
were always lower at the end sections of the pipe (Fig. S3). As stated
above, ordination of samples according to their phylogenetic related-
ness (weighted UniFrac distance, Fig. S2B) mirrored the segregation ob-
tained when samples were ordinated according to their ARGs relative
content (Fig. S2A).
3.4. Occurrence of potential pathogens in the studied sewer
OTUs obtained in the community analysis were used to investigate
the presence of potential pathogens in wastewater and biofilms by com-
paring representative OTU sequences against a database containing 283
obligate and opportunistic pathogens. This comparison yielded the oc-
currence of 69 OTUs with high sequence similarity (N98%) to well-
known bacterial pathogens. From this initial subset, 41 OTUs with rela-
tive abundances b 0.01% were discarded and only 28 were finally
retained for downstream analysis.
The most prevalent species belonged to genera Acinetobacter,
Acidovorax, Pseudomonas, Streptococcus, Clostridium, Aeromonas and,

Wastewater communities were mainly composed of members of the

phyla Bacteroidetes, Firmicutes and Proteobacteria (Fig. 3). This composi-
tion fairly agrees with previous reports on the composition of bacterial
communities in untreated sewage, which are largely dominated by bac-
terial groups more related to the sewer infrastructure than to human
feces (McLellan et al., 2010; Newton et al., 2015; Shanks et al., 2013). Re-
markably, we found a large contribution of sequences affiliated to genus
Arcobacter (class Epsilonproteobacteria) in wastewater (37.7% ± 1.68%
and 35.2% ± 0.32% of total reads in the inlet and outlet sections, respec-
tively). Despite Arcobacter species are prevalent in sewage and faecally
polluted waters (Collado and Figueras, 2011), contrasting results re-
garding their abundance in raw wastewater have been reported
(McLellan et al., 2010; Shanks et al., 2013; Tang et al., 2016; Ye and
Zhang, 2011). In our samples, the large dominance of Arcobacter-like se-
quences is of special relevance since this genus encompasses several
well-known human pathogens (e.g. A. butzleri, A. cryaerophilus and A.
skirrowii) that carry resistance genes to different antibiotics, including
penicillins and carboxypenicillins, macrolides, fluoroquinolones, Fig. 3. Average relative composition of bacterial communities in wastewater and biofilm
lincosamides and cephalosporines (Collado and Figueras, 2011; samples collected at the inlet (SP1) and outlet (SP2) sections of the sewer pipe. Values
Ferreira et al., 2015). are the mean of biological triplicates.
1052 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054

especially, Arcobacter. The relative abundance of these potential patho-
gens was then compared for wastewater and biofilm samples according
to their collection site (e.g. inlet and outlet sewer sections) (Fig. 4). Most
OTUs showing high similarity to pathogenic bacteria were significantly
more abundant at the inlet section of the sewer, especially in biofilms.
Since this inlet section directly receives raw urban sewage from the mu-
nicipality, the higher contribution of potential pathogens in this section
may reflect epidemiological patterns within the source population. In
turn, the lower contribution of pathogens at the outlet sections may
simply be the consequence of their poor survival in the sewer environ-
ment. The predominance of sequences related to A. crioaerophilus and A.
butzleri in both sewer compartments (i.e. wastewater and biofilms) at
both sections of the pipe was relevant (Fig. 4). Both species are well
recognised human and animal pathogens mainly associated with mild
and severe gastrointestinal diseases (Collado and Figueras, 2011). A
butzleri, A. cryaerophilus and, to a minor extent A. skirrowii, have fre-
quently been isolated from untreated sewage and surface waters pollut-
ed with faecal material (Collado et al., 2010, 2008; Stampi et al., 1999,
1993). The high contribution of Arcobacter-related sequences in the
studied biofilms may also be explained by their capacity to adhere to
pipe surfaces (Assanta et al., 2002). Overall, the reduced abundance of
potential pathogens at the outlet section of the sewer fairly agrees
with the concomitant decrease in the relative abundance of ARGs mea-
sured in biofilms collected at this part of the sewer. Despite this
potential linkage, further studies are needed to unequivocally associate
the observed resistance gene pool to sewage-derived bacterial patho-
gens inhabiting sewer biofilms. This observation may have, however,
major implications on the efficiency of downstream wastewater treat-
ment, the maintenance of the sewer facility and human health.
3.5. Environmental implications
Combined sewer systems convey wastewater to WWTPs where,
after treatment, it is released into the environment. Although the effec-
tiveness in the removal of antibiotics, ARBs and ARGs by WWTPs is
highly variable, wastewater treatment largely reduce nutrient content
and bacterial loads (of both harmless and pathogenic microorganisms)
to acceptable levels before discharge into aquatic environments without
risk for human and environmental health (Hendricks and Pool, 2012).
There are some scenarios, however, where untreated sewage could be
directly released into the environment. For instance, the capacity of
combined sewer systems may be exceeded during periods of heavy
rainfall thus producing sewer overflows (Murla et al., 2016). Under
these situations, a mixture of raw sewage and stormwater is directly re-
leased into the environment thus causing pollution of watercourse
(Czekalski et al., 2014). It is still unclear, however, how ARB, ARGs and
potential pathogens behave under sewer overflow conditions but it
seems evident that such situations may pose a risk to human and

Fig. 4. Comparison of the relative abundance (% of total reads) of potential pathogenic OTUs from wastewater (left panel) and biofilms (right panel) collected from the inlet (SP1) and the
outlet (SP2) sections of the studied sewer. OTUs showing significant differences in their relative abundance between inlet and outlet sewer sections are indicated (*p b 0.05; **p b 0.01). The
size of the circles next to labels varies according to sequence identity in BLAST. Putative pathogenic bacterial OTUs are ordered according to their host and pathogenic potential as specified
in left labels.
 O. Auguet et al. / Science of the Total Environment 605–606 (2017) 1047–1054
environmental health. Reference values for ARB and ARGs do not yet
exist and risk assessment models greatly depend on the bacterial host
and type of resistance. For instance, if a given resistance gene is carried
by harmless environmental bacteria, the potential risk for human health
might be low in comparison to the same gene hosted by a well-known
human (or animal) pathogen. Also, the risk also depends on the poten-
tial for gene mobilization (ARGs in bacterial genomes vs. in transmissi-
ble plasmids) (Manaia, 2017). The complete understanding of the
diversity and abundance of the sewer resistome and its dynamics
needs combined efforts from both researchers and sewer managers.
This knowledge would provide valuable data to design strategies
aimed to reduce the abundance of antibiotic resistant bacteria and path-
ogens in sewer systems. This attenuation not only will reduce the risk
associated with accidental pollution of watercourses due to sewer over-
flow events, but it would also have an additional benefit in downstream
processes occurring in WWTP by alleviating their burden in influent
wastewater.
4. Conclusions
We evaluated the occurrence of antibiotic residues and antibiotic re-
sistance genes in a pressured sewer pipe. Resistance genes were present
in liquid and biofilm phase of the studied sewer at concentrations at
least as high as other compartments of the water cycle such as WWTP
and river receiving WWTP discharges. The most abundant ARGs detect-
ed in both wastewater and biofilm samples were sul1 and sul2 with
roughly 1 resistance gene for each 10 copies of 16s RNA gene. Within
the sewer, spatial variations of ARGs were detected showing a
specificity likely due to the diverse conditions occurring within the
systems. Sewers are likely hotspots for the accumulation and
dissemination of ARGs but further research is needed to establish
transfer potential, environmental and health risks and main parameters
affecting its behaviour in the context of the urban water cycle.
Acknowledgements
The authors would like to acknowledge the Consorci Costa Brava,
Empresa Mixta Aigües Costa Brava for their help in the sampling cam-
paigns, the projects of SEWAGENE CTM2016-75653-R and REaCH
CTM2015-66892-R funded by the Spanish Government. This work has
been co-financed by the European Union through the European Region-
al Development Fund (ERDF) and supported by the Generalitat de Cata-
lunya (Consolidated Research Group: Catalan Institute for Water
Research 2014 SGR 291). S. Rodriguez-Mozaz acknowledges the Ramon
y Cajal program (RYC-2014-16707). XT was supported by project
BRIDGES CGL2015-69043-P from the Spanish Office for Research
MINECO.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.06.153.
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