Science of the Total Environment 646 (2019) 1204–1210

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Science of the Total Environment

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Impact of industrial wastewater on the dynamics of antibiotic resistance

genes in a full-scale urban wastewater treatment plant
Antonino Fiorentino a,b,1,2, Andrea Di Cesare a,c,1,3, Ester M. Eckert a, Luigi Rizzo b, Diego Fontaneto a,
Ying Yang d, Gianluca Corno a,⁎
a
Microbial Ecology Group, National Research Council of Italy, Institute of Ecosystem Study, Largo Tonolli 50, 28922 Verbania, Italy
b
Department of Civil Engineering, University of Salerno, 84084 Fisciano, SA, Italy
c
Department of Earth, Environmental and Life Sciences, University of Genoa, Genoa, Italy
d
South China Sea Resource Exploitation and Protection Collaborative Innovation Center, School of Marine Sciences, Sun Yat-sen University, Guangzhou, 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

• The impact of industrial wastewaters on

the ARGs removal in UWTPs is unclear.
• A full scale UWTP was run with or with-
out industrial wastewater for a week.
• The origin of the wastewater strongly
affects the chemical quality of a WWTP
effluent.
• Wastewater origin has a limited impact
on microbiological and genetic variables.
• PAA disinfection is not effective in the
removal of ARGs.

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

Article history: Urban Wastewater Treatment Plants (UWTPs) treating mixed urban sewage and industrial wastewater are
Received 10 May 2018 among the major hotspots for the spread of Antibiotic Resistance Genes (ARGs) into the environment. This
Received in revised form 26 July 2018 study addresses the impact of the wastewater origin on ARG dynamics in a full-scale UWTP (15,000 Population
Accepted 26 July 2018
Equivalent, PE) by operating the plant with and without industrial wastewater. Composite samples (4 L) from
Available online 27 July 2018
different treatment points were characterized for their chemical composition, bacterial abundance and for the
Editor: D. Barcelo abundance of four resistance genes against tetracycline, sulfonamides, erythromycin, and quinolones (tetA,
sul2, ermB, and qnrS), and of the class 1 integrons (intI1). Although the chemical composition of the outflow sig-
Keywords: nificantly differed when the plant operated with or without industrial wastewater, the system efficiency in the
Urban sewage removal of bacterial cells, ARGs, and intI1 was constant. The final disinfection by peracetic acid (PAA) did not af-
Industrial wastewater fect the removal of ARGs, independently of the wastewater origin and the chemical characteristics of the inflows.
Antibiotic resistance genes Our results demonstrated that a well-functioning small size UWTP could treat a significant amount of industrial
Class 1 integron wastewater mixed in the urban sewage without affecting the overall ARGs and class 1 integrons released into the
environment.
© 2018 Published by Elsevier B.V.

⁎ Corresponding author.
E-mail address: g.corno@ise.cnr.it (G. Corno).
1
These authors equally contributed to this work.
2
Present address: Department of Civil Engineering, University of Salerno, 84084 Fisciano (SA), Italy.
3
Present address: Department of Earth, Environmental and Life Sciences, University of Genoa, Genoa, Italy.

https://doi.org/10.1016/j.scitotenv.2018.07.370
0048-9697/© 2018 Published by Elsevier B.V.
 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210 1205

1. Introduction mixed), and (ii) under ad-hoc conditions (only urban wastewater). The
composite samples were characterized for their chemical composition,
The misuse of antibiotics in human and animal healthcare and in ag- for the abundance of four different ARGs: tetA, sul2, ermB, qnrS (against
riculture induces selection of bacteria carrying Antibiotic Resistance tetracycline, sulfonamides, erythromycin, and quinolones, respec-
Genes (ARGs), which unavoidably reach the environment via treated tively), and for class 1 integrons (targeting intI1 gene). These genes
and untreated wastewaters (Varela et al., 2014; Vaz-Moreira et al., were selected because they have been (i) previously detected within
2014). In particular, Urban Wastewater Treatment Plants (UWTPs) are the microbial community of the tested UWTP (Di Cesare et al., 2016a;
considered among the main hotspots for the spread of ARGs into the en- Di Cesare et al., 2016c), and (ii) recently suggested as a proxy for
vironment, since these were not designed to remove antibiotic resis- ARGs as well as of general anthropogenic contamination (Gillings
tance determinants (resistant bacteria and ARGs) from their effluents et al., 2015; Ma et al., 2017; Eckert et al., 2018).
(Rizzo et al., 2013; Guo et al., 2015).
Biological treatment in UWTPs creates a suitable environment for 2. Material and Methods
bacterial growth. Further, it might promote Horizontal Gene Transfer
(HGT) by increasing cell proximity. Flocculation and consequent biofilm 2.1. Wastewater sampling
formation might potentially increase the overall number of ARGs
(Zhang et al., 2009; Mao et al., 2015). On the other hand, efficient bio- The investigated UWTP is located in Cannobio, Piedmont Region
logical treatments with long retention time and limited nutrient load (Italy) (46°06′67″ 08°70′29″) and it serves 15,000 Population Equiva-
could favor a rapid shift towards a typical aquatic microbial community, lent, with an average flowrate of 3500 m3 day−1, and a flow peak
reducing the proportion of bacteria carrying ARGs and of human patho- (15% increase) typically occurring in the early morning and evening
gens by the concomitant effect of different ecological interactions (com- hours. The UWTP receives urban wastewater from the town of Cannobio
petition and predation) (Di Cesare et al., 2016a). (about 7000 inhabitants in winter, no large healthcare facilities)
Recent studies show that disinfection processes, the last step in through the sewage system and receives industrial wastewater from
wastewater treatment in several countries, are efficient in the removal
of residual bacterial cells, but select for ARGs (Di Cesare et al., 2016a;
Alexander et al., 2016). In a few cases, the number of ARGs in the efflu-
ent of UWTPs even increased compared to the influent (Czekalski et al.,
2012; Lee et al., 2016).
Several aspects of antibiotic resistance in wastewater have been in-
vestigated, in particular (i) ARG dynamics within microbial communi-
ties in UWTPs (Mao et al., 2015; Guo et al., 2017), (ii) the fate of ARGs
once released into the environment (Czekalski et al., 2014; Subirats
et al., 2017), (iii) the defense strategies promoted in antibiotic resistant
bacteria (ARB) during the wastewater treatments (Turolla et al., 2017),
and (iv) the enrichment of some ARGs in the presence of heavy metals
(Mao et al., 2015). However, the fate of ARGs when mixing industrial
and urban wastewater, a common occurrence in many UWTPs, is an un-
explored black box. In general, the impact of chemical and biological pa-
rameters on antibiotic resistance under operational conditions, with or
without additional inputs of industrial wastewater, has not been
addressed.
The effect of wastewater (urban or industrial) on ARG dynamics was
compared between two different UWTPs, one treating urban and poul-
try farm wastewater, and one that receives urban and industrial waste-
water (Lee et al., 2017). A study on the same UWTP operated at full scale
with and without industrial wastewater has not been conducted. As a
common practice, several UWTPs also receive (by tankers) industrial
wastewater from other facilities (e.g., leachate from landfills, specific
wastewater from factories, etc.), which are stored in silos before being
treated. These wastewaters, generally contaminated by toxic organic
and inorganic pollutants (e.g., N-based compounds and heavy metals),
are then mixed with urban wastewater coming from sewage in a dilu-
tion ratio sufficient to avoid any adverse effect on the biological water
treatment process (Mikosz, 2015). The different chemical composition
of urban and industrial wastewaters could affect the overall ARG profile
within the microbial communities in UWTPs, in particular because of
the occurrence of high concentrations of heavy metals. The selection
of bacteria resistant to metals, thus carrying specific metal resistance
genes, is directly linked to potential co-selection of ARGs within stressed
microbial communities (Di Cesare et al., 2016b). A strong correlation be-
tween ARGs and metal resistance genes was observed within the micro-
bial communities of different UWTPs (Di Cesare et al., 2016c). Fig. 1. Chemical composition of the wastewater. Dendrogram of complete linkage
There is a need to better understand the fate of the antibiotic clustering of Euclidean distance between the different samples in terms of absolute con-
resistome (all ARGs and their abundances within a bacterial commu- centrations of chemical parameters, associated with heat map of relative distribution of
chemical parameters in the various treatments in WWTP fed with and without industrial
nity) in UWTPs treating industrial wastewaters. In this study, the effi- wastewater. On the vertical column on the left, the measured chemical parameters are
ciency of removing ARGs in a full-scale UWTP was investigated listed. Different symbols refer to different sampling points along the water treatment
(i) under typical operating conditions (urban and industrial wastewater and the normalized relative abundance is defined through the scale of greys reported.
1206 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210

Table 1 wastewater (from now UW) from Cannobio sewage system. The second
Chemical composition of wastewaters in the UWTP of Cannobio. The UWTP was sampled sampling campaign was carried out 7 days after the first one, in order to
along the whole treatment process in the different operating conditions (UIW and UW).
operate for a week the UWTP with urban wastewater only to minimize
UIW UW possible residual effect of industrial wastewater. Weather conditions
Inflow Post Post Outflow Inflow Post Post Outflow were characterized by high atmospheric pressure, with no precipitation,
biol. chem. biol. chem. storm flows, nor snow melts, in the week before the first sampling cam-
COD mg/l 294 181 83 101 345 139 52 69 paign and between the two sampling campaigns. Samples were manu-
BOD5 mg/l 205 92 16 12 210 45 b5 10 ally collected every 3 h starting at 08:00 AM for 24 h, and kept at 4 °C in
Al mg/l 0.34 1.70 0.54 0.50 0.33 2.00 0.40 0.42 the dark until further analyses (performed within the next 8 h). Taking
Sb μg/l 0.0 2.0 1.0 1.0 b0.3 4.0 3.0 3.0 into account the retention time of the WWTP (~10 h for waters, ~8 days
Ba μg/l 35.0 48.0 31.0 53.0 41.0 46.0 59.0 46.0
B mg/l 0.19 1.17 1.22 1.23 0.10 0.80 0.78 0.81
for sludge), one-day integrated samples as previously described (Di
Ca mg/l 26.8 30.9 22.5 23.0 33.8 42.6 36.7 37.2 Cesare et al., 2016a) were produced for each sampling point from
Co μg/l 0.32 2.00 2.00 2.00 0.34 9.00 7.00 7.00 pooling equal amounts (500 mL) of each 3 h sample for a final volume
Cu mg/l 0.11 0.07 0.00 0.00 0.11 0.07 0.01 0.01 of 4 L. The samples were collected in: (i) the inflow before the mechan-
Fe mg/l 0.51 0.38 0.09 0.09 0.42 0.28 0.04 0.04
ical treatment, (ii) the effluent of the secondary sedimentation tank
Pb mg/l 0.007 0.009 0.004 0.011 0.012 0.011 0.012 0.010
Mg mg/l 5.02 5.54 3.89 3.90 6.75 6.97 5.27 5.36 (post biological treatment), (iii) the effluent of the chemical treatment
Mn mg/l 0.03 0.05 0.04 0.04 0.03 0.07 0.06 0.06 (post-chemical), and (iv) the effluent of the disinfection process (out-
Hg μg/l 2.0 4.0 3.0 2.0 1 5.0 3.0 2.0 flow). Each one-day integrated sample was pre-filtered over a 126-μm
Mo μg/l 1.0 17.0 14.0 13.0 2 45.0 41.0 41.0 pore-size membrane (Sartorius AG, Germany) to remove large particles.
Ni mg/l 0.004 0.157 0.129 0.127 0.005 0.156 0.126 0.131
K mg/l 8.69 31.27 31.20 30.51 11.13 89.63 83.59 85.69
Aliquots of 2 mL of the ultrafiltrate were then fixed in formaldehyde (1%
Ag μg/l 0.32 1 0.24 b0.1 0.26 0.44 0.20 0.11 final concentration) for cytometric analyses of the bacterial community,
Na mg/l 28.6 315.0 309.0 311.0 36.5 470.9 419.7 433.5 aliquots of 2 L were used for chemical analyses, and the remaining sam-
Th μg/l 1.00 0.25 0.17 b0.1 0.14 0.11 b0.1 b0.1 ple, approximately 0.2–0.8 L, was collected on 47 mm diameter, 0.22 μm
Total mg/l 0.002 0.027 0.012 0.013 0.002 0.028 0.012 0.013
pore-size nitrocellulose filters (Nucleopore, Merck Millipore Co.,
Cr
U μg/l 4.0 2.0 2.0 3.0 5.0 2.0 2.0 3.0 Germany) and stored at −20 °C until DNA extraction.
Zn mg/l 0.250 0.135 0.067 0.066 0.135 0.144 0.070 0.074
2.2. Chemical characterization of the wastewater

tankers, at a proportion of 30–40% in volume to the overall inflow. In
particular, industrial wastewater includes landfill leachate (European
Waste Catalogue (EWC) code EWC 190703) and non-hazardous aque-
ous liquid wastes (EWC 161002). The treatment includes (i) a mechan-
ical separation of solids followed by (ii) a conventional biological
treatment (activated sludge with a sludge retention time of 8 days) op-
erated in pre-denitrification mode (for nitrogen removal) with a sec-
ondary sedimentation, (iii) a chemical treatment (aluminum
polychloride enriched by sodium hydroxide), and (iv) a final disinfec-
tion by peracetic acid (PAA) (for a graphical depiction of UWTP treat-
ment scheme see Supplementary Figure SF1). The industrial
wastewater, pretreated with a classical Fenton process, is continuously
added in the denitrification tank. The industrial wastewater is a mixture
of (i) chemically and physically pre-treated industrial waste, and (ii)
leachate from urban solid waste landfills. Samples were collected on
February 14–15, 2017, with the UWTP operated under typical condi-
tions with mixed urban/industrial wastewaters (from now mentioned
as UIW). The inflow from industrial wastewater was then closed imme-
diately after sampling, and on February 22–23, 2017 samples were col-
lected again, when the UWTP was operated only with urban
Chemical analyses were performed by the Acqua Novara.VCO labo-
ratory (Dormelletto, Italy). The pre-filtered integrated samples were
immediately processed. In detail, the concentrations of Al, Ag, B, Ca, Cr,
Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn were measured according to Stan-
dard Methods and QA/QC procedures (APHA et al., 2012) by using an
ICP-OES (by Perkin Elmer® Optima™ 8000, with the exception of B
and V, measured with a Perkin Elmer® Optima™ 2100). The concentra-
tions of Ag, Ba, Co, Hg, Mo, Sb, Se, Th, and U were measured according to
the National Environmental Protection Agency EPA Method 200.8
(Environmental Protection Agency (U.S.E.P.A.), 1994) by using an
Agilent IGC/MS.
COD (chemical oxygen demand) (mg O2/L) and biological oxygen
demand (BOD5) (mg O2/L), expressed as acetate equivalent, were deter-
mined using LCK HACH Lange cuvettes, according to the standard ISO
6060-1989 DIN 38409-H41-H44 and DIN EN 1899-1, respectively.
2.3. Bacterial abundance and size distribution
Aliquots of 2 mL from each integrated sample were used to measure
bacterial variables (abundance and size distribution) by flow cytometry

108 108
Dilution effect A B

Dilution effect
aggregates ml

107 107
-1
cells ml

-1

Difference in efficiency
106
Difference in efficiency 106

Inflow Post-bio Post-chem Outflow Inflow Post-bio Post-chem Outflow

Fig. 2. Bacterial (A) and aggregate (B) abundances. Data refers to the different sampling points during the treatment of UIW (white circles) and UW (black circles). Data are presented as
mean ± standard deviation.
 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210 1207

(Accuri C6, BD Biosciences) and, in case of unclear cytometric outputs,
by epifluorescence microscopy (Axioplan, Zeiss). Aliquots of 0.5 mL
were stained with SYBR Green I (ThermoScientific Inc.) solution (1%)
for 15 mins in the dark and then processed in the cytometer, by setting
in the cytograms a minimum of 2 × 106 events within the gate set for
single bacterial cells, and 5 × 102 events in the gate set for N3 cells
clusters (Corno et al., 2013). Six randomly selected counts were checked
for accuracy by epifluorescence microscopy (DAPI stained bacterial
community collected on 0.2 μm polycarbonate filters, Millipore). Bacte-
rial aggregates from those samples were carefully checked at the micro-
scope before being assigned to the correspondent gate in the cytograms.
According to previous studies on this UWTP, the largest majority of

Fig. 3. intI1 and single ARGs abundances. Genes are quantified for each different sampling point in UIW (white symbols) and UW (black symbols) (expressed as gene copies mL−1)
depicted as average of three measurements ± standard deviation.
1208 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210

Fig. 4. ARG distribution within the wastewater. Dendrogram of complete linkage clustering of Euclidean distance between the different samples in terms of absolute abundances of an-
tibiotic resistance genes.

aggregates were solely composed by bacterial cells, resulting either intI1 were expressed as gene copies mL−1 and as gene copy on
from the subdivision of a single initial cell (microcolonies) or from me- 16SrRNA copy.
chanical attachment of different cells between themselves or to small
particles of detritus. The number of predators (either heterotrophic 2.5. Statistical analysis and graphical depiction
nanoflagellates or mixotrophs) attached to the aggregates was negligi-
ble. Gates and counting per mL were done with the Accuri C6 resident All data analyses were conducted with R 3.1.2 (Team, R. C., 2017)
analysis software (BD Biosciences). using RStudio (Team, R., 2015). Figures were made in R without a spe-
cific package or with ggplot2 v1.0 (Wickham, 2009) and reshape2 v1.4
(Wickham, 2007) and were additionally processed in Adobe Illustrator
2.4. DNA extraction, 16SrRNA, antibiotic resistance genes and class 1 CS5.
integron-integrase gene (intI1) quantification Differences between samples in terms of chemical composition and
ARG profiles of each sampling day/step were calculated as Euclidean
Three quarters of each Nucleopore filter were separately processed distance between the concentration of chemical parameters and ARGs
for the DNA extraction using a commercial kit (Ultra Clean Microbial absolute abundance (ARGs mL−1) and relative abundance (ARG num-
DNA Isolation Kit, MoBio Laboratories) following the manufacturer's in- ber per 16SrRNA gene copy). For graphical depiction, samples were
structions. DNA samples were diluted 1:100 and the abundance of clustered with complete linkage. PERMANOVA analysis was conducted
16SrRNA, the different ARGs (tetA, sul2, ermB, qnrS) and of intI1 were to evaluate the importance of the sampling day, the sampling point,
assessed by quantitative PCR (qPCR) using the same primers and set- and the interaction of the two factors in explaining the clustering of
tings described previously (Di Cesare et al., 2016a). Efficiencies and R2 the distances of the samples, using the adonis command in the package
averages for the tested genes were 96.33 ± 4.43 and 0.98 ± 0.01. The vegan v2.2-1 (Oksanen et al., 2017) for relative and absolute abundance
limits of quantification, determined according to the procedure de- of ARGs including and excluding the inflow. Clustering for graphical de-
scribed by Bustin and coworkers (Bustin et al., 2009), were 2.32 × 103, piction was conducted including the inflow of urban wastewater. Single
12, 36, 54, 22, and 33 copies μL−1 for 16SrRNA, tetA, sul2, ermB, qnrS bacterial cells and cells cluster abundances (log transformed, and tested
and intI1, respectively. The abundances of the different ARGs and of for normality of the model residuals) were tested for significant differ-
ences between UIW and UW by applying a two-way repeated measures
analysis of variance (RM-ANOVA).
Table 2
Results of PERMANOVA analysis of the importance of the WWTP step, the sampling day
(UIW and UW) and their interaction on the distances between samples in terms of abso-
3. Results and discussion
lute (A. & B.) and relative (C. & D.) gene abundances: A. & C. including the samples from the
inflow and B. & D. excluding them. Highest R2 values are indicated in bold font. 3.1. Wastewater origin impact on chemical parameters
2
F model R p-Value
The chemical composition of the wastewater was highly influenced
A. Absolute ARG abundances including Inflow
by its origin, either urban + industrial (UIW) or urban only (UW)
WWTP step 65.828 0.73177 0.001
Day 15.184 0.05626 0.005 (Fig. 1, Table 1). The industrial wastewater was introduced into the sys-
Day:WWTP step 13.734 0.15268 0.008 tem directly in the pre-denitrification tank and it had a strong impact on
the chemical composition of the mixed wastewater. This difference
B. Absolute ARG abundances excluding Inflow
WWTP step 47.977 0.86818 0.001 persisted through the biological treatment and to the final effluent. On
Day 1.573 0.01424 0.299 the basis of their chemistry, all samples clustered according to the origin
Day:WWTP step 0.498 0.00901 0.644 of the wastewater, independently of treatment step (Fig. 1). Indeed, 62%
C. Relative ARG abundances including Inflow of differences between the chemical profiles of the samples, from the bi-
WWTP step 121.655 0.91629 0.001 ological process to the outflow, are explained by the difference between
Day 7.552 0.01896 0.017 UIW and UW, while the UWTP treatment steps explain only 30%
Day:WWTP step 3.264 0.02458 0.083 (Permanova). In the inflow, the concentration of COD (294 and
D. Relative ARG abundances excluding Inflow 345 mg L−1 for UIW and UW respectively) and BOD5 (205 and
WWTP step 1.6727 0.15469 0.148 210 mg L−1 for UIW and UW respectively) were very similar. However,
Day 2.9928 0.13838 0.037 the addition of industrial wastewater drastically affected the perfor-
Day:WWTP step 1.6446 0.15209 0.168
mance of the biological processes. After the biological treatment, COD
 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210
decreased to 181 mg L−1 during the UIW treatment (b40% reduction
compared to the corresponding inflow) and to 139 mg L−1 for UW
treatment (reduction from the inflow N60%). The BOD5 was reduced
after the biological treatment to 92 mg L−1 for UIW (−55%) and to
45 mg L−1 for UW (−79%). After chemical treatment and disinfection
the concentration of BOD5 was approximately 12 mg L−1 (−94%) in
UIW and around 10 mg L−1 in UW (− 96%), while the concentration
of COD was 101 mg L−1 in UIW (−66%) and 69 mg L−1 in UW (−80%).
Ibarbalz and colleagues (Ibarbalz et al., 2013) studied the profile of
the bacterial communities in seven industrial wastewater treatment
plants (IWTPs) and in one UWTP. Their work shows that each activated
sludge system in the IWTPs exhibited a unique bacterial community,
which was clearly different from the community observed in the
UWTP. They hypothesized that the bacterial communities in activated
sludge tanks responsible for the removal of BOD are more diverse in
the IWTPs, and this could lower the process kinetics. Wastewater treat-
ment plants are commonly designed with default parameters set for
municipal sewages (Insel et al., 2012), but when the UWTP are designed
to also treat industrial wastewater, engineers expect a lower removal ki-
netics in the active sludge tank (Stricker and Racault, 2005; Çokgör et al.,
2009). Also in our work, when urban sewage was mixed with industrial
wastewater, the inactivation kinetics of BOD and COD were lower in
comparison to those of the solely urban wastewater treatment.
Examining metal composition, from the post biological tank for-
ward, the concentration of metals, with the exception of some (i.e., Al,
Ba, Cr, Cu, Pb, Hg, Ni, U and Zn), was generally different in UIW and
UW (Fig. 1). Ag, B, Fe, and Th had higher concentrations following the
addition of the industrial wastewater. In particular, Ag concentration
in the post-biological treatment doubled (1.00 mg L−1 versus
0.44 mg L−1), B concentration is of 1.20 mg L−1 in UIW and
0.78 mg L−1 in UW (means of post-biological, −chemical, and outflow).
Also Fe and Th concentrations were higher in UIW than in the UW, es-
pecially in the post-biological treatment (33% and 228% more respec-
tively). These metals commonly characterize landfill leachates (Dolar
et al., 2016), which constitutes an important portion of industrial waste-
water treated in the UWTP of Cannobio. Moreover, other heavy metals
are also present in leachates and, in general, their concentration de-
pends on the age of the landfill (Kulikowska and Klimiuk, 2008; Foo
and Hameed, 2009). In contrast, Ca, K, Mg, Mn, Na, Co, Mo, and Sb con-
centrations were much higher in the UW than in the UIW. In the UWTP
of Cannobio the most prominent differences were detected for Co, Mo,
and Sb whose concentrations in the three treatment steps were about
three times higher in the UW treatment (Table 1). The presence of Co
and Mo in higher concentrations in the UW can be linked to the pres-
ence of these elements in products of domestic use, including plant
care products, nutritional supplements, and household cleaning prod-
ucts (Department of Health and Human Services, 2017). The presence
of Sb in higher concentrations in the UW could be due to the presence
of this metal in the joints of domestic pipes (Department of Health
and Human Services, 2017). Ca, K, Mg, and Na are present in almost
all the foods in the Italian diet (European Institute of Oncology (IEO),
2017) and therefore it was normal to expect their highest concentration
within the only UW inflow. Overall, these results show that the intro-
duction of industrial wastewater has a strong impact on the chemical
composition and especially on the abundance of the different metals
within the UWTP, accordingly to the established knowledge (Mantis
et al., 2005).
3.2. Bacterial community and ARGs distribution analyses
Bacterial single cells number decreased from the inflow to the final
outflow by 1.5 log units (in particular, a decrease of 0.8 log units was
found in the post-biological and 0.5 log units in the post-chemical sam-
ples). A comparable trend was detected for microbial aggregates (1.0
log units overall decrease, 0.3 log units for UW and 0.8 for UIW in the
post-biological, 0.5 for UW and 0.1 for UIW in post-chemical) (Fig. 2).
1209
The highest decrease was thus detected after the biological treatment
for both single cells and aggregates. Only a slight decrease was observed
after the tertiary treatment, and even the disinfection step poorly af-
fected both parameters, supporting previous results from UWTPs in
Cannobio and other Italian treatment plants (Di Cesare et al., 2016a;
Di Cesare et al., 2016c). Despite the general dynamic towards reduction,
which is very similar for the two types of wastewater, bacterial single
cells and aggregates were always slightly but significantly lower in
UIW compared to the respective UW samples (RM-ANOVA: p = 0.033
for single cells; p = 0.050 for aggregates). This difference increased in
the post-biological samples, especially for the aggregates (p = 0.015
for single cells, p = 0.002 for aggregates). Aggregation is a typical re-
sponse of bacteria to stress, e.g. in response to antibiotics (Corno et al.,
2014) or other toxins, predation or competition for nutrients (Corno
and Jürgens, 2008). In this case, however, the difference in chemical
composition of the industrial wastewater and the effect of dilution on
the overall bacterial number had a limited effect on bacterial aggrega-
tion, while the dilution implemented in UIW, reducing cells density,
possibly reduced the chances for bacteria to mechanically meet and
grow in clusters. As expected, ARG and intI1 abundance followed a sim-
ilar dynamic of reduction in absolute numbers as bacterial cells and ag-
gregates (Figs. 2, 3). Furthermore, analyzing the whole treatment
process, starting from the post-biological step (where industrial wastes
were added) to the effluent after the final disinfection, both classes of
the tested genes varied following similar trends, independently of the
contribution of the industrial wastes, and without notable differences
between single genes (Fig. 3). The overall removal efficiencies in
terms of absolute gene concentrations were 9, 10, 14, 8, and 8% for
intI1, tetA, sul2, qnrS, and ermB respectively. This result shows that the
introduction of industrial wastewater did not affect the process in
terms of removal of ARGs and class 1 integrons. This result reinforces
the possibility to use the intI1 gene as proxy of ARGs dynamics in waste-
water and the environment (Ma et al., 2017; Eckert et al., 2018).
UWTP samples collected within the water treatment clustered in
two groups: one containing all post-biological treatment samples and
another one containing all post-chemical and final outflow samples,
suggesting that the type of wastewater (UIW or UW) does not affect
the abundance of ARGs and of the class 1 integrons (Figs. 3, 4). Indeed,
excluding the inflows for which the sampling day has a slight effect,
86% of the variance is explained by the treatment for absolute gene
abundances (Table 2). In terms of relative gene abundances (Supple-
mentary Figure SF2) the removal was influenced by only 15 and 14%
by the treatment and day, respectively, thus none of the two factors
was very important if the inflow is excluded (Table 2). On the contrary,
including the inflow, the treatment explains 92% of the variance of the
relative gene abundances (Table 2). Lee and colleagues (Lee et al.,
2017) showed a clear difference in the removal (or towards a selection)
of specific ARGs was observed comparing two different UWTPs, fed with
livestock or industrial wastewater together with urban wastewater.
Their research was limited by the different design and the different effi-
ciency of the two analyzed treatment plants. Our study is the first car-
ried out on a full-scale UWTP typically fed with a mixture of urban
and industrial wastewater, by stopping the industrial feeding for one
week before the UW sampling. The results indicate that the type of
the wastewater (namely UIW vs UW) affects the chemical composition,
and the overall bacterial number and clustering behavior, but does not
affect the investigated ARGs and intI1 relative abundances within the
resident microbial communities. The disinfection step did not affect
the removal of ARGs, independently of the source and the chemical
characteristics of the inflows. Therefore, disinfection by PAA is not an ef-
fective barrier for controlling the release, and possible spread into the
environment, of antibiotic resistance. Our results highlight the impor-
tance of an efficient biological treatment within UWTP. This step is crit-
ical in determining the composition of the microbial community of the
final effluents, and thus the fate of antibiotic resistance determinants
in the environment. Once the biological treatment effectively works,
1210 A. Fiorentino et al. / Science of the Total Environment 646 (2019) 1204–1210
the reduction in bacterial numbers and in ARGs becomes relatively in-
dependent of the quality of the inflow.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.07.370.
Competing interests
The authors declare that no competing interests exist.
Acknowledgments
We thank Dr. Alessandro Garavaglia, Mr. Arnaldo Buttignol, and the
staff of the UWTP of Cannobio (Acqua Novara.VCO S.p.A.) for
performing the chemical analyses, for providing full access and free ma-
nipulation of the treatment processes, and for the support in the sam-
pling. This research is supported by Acqua Novara.VCO S.p.A. and by
the Bilateral High Relevance Project Italy-China nr. CN16GR05 “Effi-
ciency of different disinfection processes in the removal of antibiotic re-
sistance determinants in experimental pilot systems and full-scale
WWTPs”.
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