Environmental Science and Pollution Research (2023) 30:35294–35306

https://doi.org/10.1007/s11356-022-24650-w

RESEARCH ARTICLE

Occurrence of antibiotic‑resistant bacteria and resistance genes

in the urban water cycle
Silvia Bonetta1 · Andrea Di Cesare2 · Cristina Pignata3 · Raffaella Sabatino2 · Manuela Macrì1 · Gianluca Corno2 ·
Marco Panizzolo3 · Sara Bonetta3 · Elisabetta Carraro3

Received: 2 September 2022 / Accepted: 3 December 2022 / Published online: 17 December 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
This study investigates the antibiotic resistance fate in the urban water cycle, evaluating the dynamics of antibiotic-resistant
bacteria (ARB) and antibiotic-resistant genes (ARGs) in three different full-scale wastewater treatment plants (WWTPs)
and two drinking water treatment plants (DWTPs) located in the same geographical area (North-West of Italy). ARB (tetra-
cycline-, ampicillin-, and sulfonamide-resistant bacteria) were quantified by plate counting and the abundances of selected
ARGs (i.e., tetA, blaTEM, and sulII) and intI1 gene were measured using quantitative real-time PCR (qPCR). Higher con-
centrations of ARB and ARGs were observed in the WWTPs with respect to the DWTPs identifying the WWTP as hotspot
for the spread of antibiotic resistances. Although a significant reduction of ARB and ARGs was observed in WWTPs and
DWTPs after the treatment, none of the detected ARB or ARGs was completely removed in drinking water. The stability of
the antibiotic-resistant rates between inlet and outlet associated with the reduction of relative ARG abundances underlined
that both the treatments (WWTs and DWTs) did not apply any selective pressure. The overall results highlighted the impor-
tance to investigate the antibiotic resistance dynamics in aquatic ecosystems involved in urban water cycle integrating the
information obtained by culture-dependent method with the culture-independent one and the need to monitor the presence
of ARB and ARGs mainly in drinking water that represents a potential route of transmission to human.

Keywords Urban water cycle · Antibiotic resistance · Antibiotic resistance bacteria · Antibiotic resistance genes ·
Wastewater treatment · Drinking water treatment

Introduction interventions are not carried out to overcome infections

attributable to microorganisms resistant to antimicrobials,
Antimicrobial resistance is of primary concern for the public there could be an increase in deaths estimated up to 10 mil-
health (WHO 2014). Globally, it is estimated that 700,000 lion people in the world by 2050, each year (O’Neil 2016).
people each year could die because of antimicrobial-resistant The main driver for the spread and the persistence of anti-
bacterial infections (Carvalho and Santos 2016). If effective biotic resistance is the overuse and misuse of antibiotics
in human and animal medicine (Sanganyado and Gwenzi
2019). Antibiotic-resistant bacteria and resistance genes
Responsible Editor: Robert Duran. (ARB and ARGs) together with residues of antibiotics are
released in sewer that constitutes a reservoir of ARB and
* Silvia Bonetta ARGs (Rizzo et al. 2013).
[email protected]
In this context, waters, favoring dispersion and smoothing
1
Department of Life Sciences and Systems Biology, physical factors (e.g. temperature, UV radiation) reducing
University of Torino, Via Accademia Albertina 13, the survival rates of allochthonous bacteria in the environ-
10123 Torino, Italy ment, allow the spread of human- and animal-derived bac-
2
Molecular Ecology Group (MEG), National Research teria; therefore, it plays an important role in the release of
Council of Italy – Water Research Institute (CNR-IRSA), antibiotic resistances into the environment. In particular, the
Largo Tonolli 50, 28922 Verbania, Italy
urban water cycle that includes the collection, treatment, and
3
Department of Public Health and Pediatrics, University discharge of treated wastewater into the environment as well
of Torino, Piazza Polonia 94, 10126 Torino, Italy

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as the abstraction, disinfection, and distribution of water for reach the final consumer of the water (Manaia et al. 2016).
drinking purposes represents an interesting model for tracing Although the direct impact on human health by ARB and
the fate of antibiotic resistance in the environment and for ARGs in drinking water is not well established, antibiotic
assessing the risk of transmission to humans (Manaia et al. resistance threats human health by two main different mech-
2016; Almakki et al. 2019). anisms: first, pathogenic and opportunistic ARB that can
In the last two decades, several studies reported the survive in drinking water may also enter the human micro-
presence of ARB and ARGs in different concentrations biome following water consumption (Vaz-Moreira et al.
in aquatic environments all over the world (Baquero et al. 2014); second, ARGs in drinking water can occasionally
2008; Li et al. 2015; Yang et al. 2019). In particular, urban be transferred by horizontal gene transfer (HGT) to human
wastewater treatment plants (WWTPs) are among the most pathogenic bacteria (Manaia 2017). Therefore, drinking
important receptors and hotspots for the release of antibiotic water can be considered as a potential transmission route of
resistance into the environment (Rizzo et al. 2013). A recent antibiotic resistance to humans (Chang et al. 2015; Manaia
overview on ARG occurrence in WWTPs highlights that the et al. 2016).
absolute abundance of the most frequently detected ARGs Although drinking water treatment drastically abates the
in influent worldwide ranged from 4.5 to 7 log copies/mL overall bacterial numbers, including the number of ARB
(Wang et al. 2020). Also antibiotic-resistant fecal indicators and ARGs, still a small but quantifiable number of cells
(Escherichia coli, coliforms) or ARB isolated from hetero- and genes have been detected in several drinking water sys-
trophic flora were frequently reported in WWTP influents, tems. Indeed, the standard drinking water treatment plants
with a mean concentration of 4 and 6 log of colony-forming (DWTPs) are not specifically designed to reduce ARB and
units (CFU)/mL detected in different studies for tetracycline- ARGs (Huang et al. 2021). Moreover, biofilm formation
and sulfonamide-resistant bacteria, respectively (Munir et al. (Zhang et al. 2019), presence of chlorine residues (Bai et al.
2011; Gao et al. 2012). 2015), and heavy metals allow the persistence of antibiotic
Despite the wastewater treatments generally lead to a resistances in drinking water systems (Seiler and Berendonk
reduction of ARG and ARB abundance (~ 2–3 logs), some 2012).
studies observed higher resistance rates in the effluents with This study investigates the antibiotic resistance fate in
respect to the influent, in relation to the different treatment the urban water cycle, evaluating the dynamics of ARB and
processes investigated (Pazda et al. 2019; Wang et al. 2020; ARGs in three different full-scale WWTPs and two DWTPs
Stachurova et al. 2021). located in the North-West of Italy.
Thus, it is important to underline that even a well-func- The combination of culture-dependent and culture-
tioning WWTP equipped with secondary and even tertiary independent approach was used. In particular, tetracycline-,
treatment will be able to release high concentrations of ARB ampicillin-, and sulfonamide-resistant bacteria in the influ-
and ARGs into the environment. Some studies reported ent and in the final effluent of each WWTP and DWTP were
that, in a final effluent of a WWTP, it is possible to detect quantified by plate counting. Moreover, the abundances of
about ­109–1012 CFU of total bacteria per day per inhabitant selected ARGs (i.e., tetA, blaTEM, and sulII; against tetracy-
equivalent; of these, at least ­107–1010 showed some form of cline, β-lactams, and sulfonamides, respectively) as well as
antibiotic resistance (Rizzo et al. 2013). Moreover, a recently those of the integrase gene of the class 1 integrons (intI1),
pan-European survey on treated wastewater demonstrates used as proxy of the anthropogenic pollution and of the anti-
that WWTPs are responsible for the discharge of consid- biotic resistance in the environment (Gillings et al. 2015;
erable amounts of ARGs in the downstream water bodies Ma et al. 2017), were measured using quantitative real-time
(Cacace et al. 2019). These data underline the main role of PCR (qPCR).
WWTPs in the accumulation and spreading of ARB and
ARGs into open waters.
Several studies evaluated the antibiotic resistance in Materials and methods
WWTPs worldwide observing highly variable abundances
of ARB and ARGs; these results suggested the influence of Sampling
local environmental and anthropogenic factors (e.g., anti-
microbial residue levels, bacterial taxonomic composition, WWTPs
local use of antimicrobials) and they highlighted the need
to specifically evaluate antibiotic resistance in different geo- Influent and effluent samples were collected in three
graphical areas (Hendriksen et al. 2019). WWTPs (plants A, B, and C) located in North-West of Italy.
The investigation of antibiotic resistance in the urban The WWTP A (population equivalent of 3,800,000) employs
water cycle represents a relevant issue considering the risk preliminary treatment (screening and aerated grit removal),
that ARB and ARGs in the “unclean” phases of the cycle can primary sedimentation, biological process (denitrification/

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oxidation/nitrification), secondary settling, then phos- of drinking water (0.5–1 L for HPC and 2 L for ARB) were
phorus removal and filtration steps (multilayer anthracite/ filtered (0.22-µm pore nitrocellulose size filter membrane,
sand beds). The WWTPs B and C (60,000 and 276,000 Millipore) in duplicate and then the membranes were placed
population equivalent, respectively) after screening and grit on ­R2Agar plates with and without antibiotics.
removal employ a denitrification step, the biological treat- All plates were incubated at 30 °C for 7 days and the
ment, and the secondary settling. In addition, the WWTP B results are expressed as log CFU/mL. The antibiotic resist-
has a primary sedimentation step. Finally, a tertiary treat- ance rate for each antibiotic was calculated as the ratio
ment is carried out with ultrafiltration in WWTP B and with between the CFU/mL of each ARB and the CFU/mL of HPC.
a chlorination step in WWTP C.
Six wastewater samplings were performed during 1 year Sample processing and DNA extraction
(March 2019–January 2020). One sample (2 L) was taken
in each sampling point of the WWTPs. Samples collected in Samples of wastewater influent (20 mL), wastewater efflu-
sterile plastic bottles were transported on ice to the labora- ent (250 mL), drinking water source (700 mL), and finished
tory and analyzed within 24 h. water (3 L) were filtered in triplicate on 0.22-µm pore size
polycarbonate filter membrane. The filters were stored
DWTPs at − 20 °C until DNA extraction. Subsequently, the entire
filters were processed for the DNA extraction using the
Drinking water source (raw surface water from a large DNeasy PowerWater kit (Qiagen) according to the manu-
river) and finished water (drinking water) samples were facturer’s instructions. Concentration of the extracted DNA
collected in two DWTPs (plants D and E) in the same of each sample was quantified by spectrophotometry (Nan-
geographical area of WWTPs (~ 20 ­km2 in North-West of oDrop® ND-1000, NanoDrop Technologies, Wilmington,
Italy). Plants D and E treat 130,000 m ­ 3/day and 86,400 m­ 3/ DE).
day of drinking water, respectively, provided by the same
drinking water source. Real‑time qPCR of ARGs
The DWTP D involves pre-decantation, addition of pow-
der-activated carbon (PAC), ozonation followed by clari- The abundance of the selected ARGs (tetA, blaTEM, and sulII)
fication/flocculation, two granular-activated carbon (GAC) of intI1 and of the 16S rRNA gene was measured by qPCR
filtrations, and final disinfection using chlorine dioxide. The using a RT-thermocycler (CFX Connect, Bio-Rad). The pro-
DWTP E employs chlorination, clarification, GAC filtration, tocol used in qPCR assays was previously described by Di
and final disinfection using chlorine ­dioxide. Cesare et al. (2015). The qPCR program was 95 °C for 2 min,
Six water samplings were performed during 1 year 35 cycles of 95 °C for 15 s, annealing temperature reported
(March 2019–January 2020). One sample was taken in each in Supplementary Table S1 for 30 s, and 72 °C for 15 s. Melt
sampling point of the DWTPs. The sampling volume was 3 curve analysis was performed from 60 to 95 °C with incre-
L for raw surface water and 30 L for drinking water. Samples ments of 0.5 °C/5 s. Standard calibration curves were carried
collected in sterile plastic bottles were transported on ice to out using the purified, quantified, and ten-fold diluted ampli-
the laboratory and analyzed within 24 h. con of each gene as described in Di Cesare et al. (2013). Each
reaction was carried out in duplicate for each sample. The
Sample processing and ARB quantification limits of quantification (LOQ) per each quantified gene were
determined as described in Bustin et al. (2009). They were
ARB were isolated in a basic culture medium for the count 1.55 × ­103, 1.22 × ­102, 3.57 × ­101, 4.88 × ­102, and 1.12 × ­101
of heterotrophic bacteria ­(R2Agar, VWR International, gene copy/µL for 16S rRNA, tetA, sulII, blaTEM, and intI1
Milan) supplemented with ampicillin, tetracycline, and sul- genes respectively. The mean value ± standard deviation of the
famethoxazole at a concentration of 32 mg/L, 16 mg/L, and reaction efficiencies was 98.32 ± 8.23% and the R2 was always
50.4 mg/L, respectively. more than 0.97. The potential inhibition of the qPCRs due to
The antibiotic concentrations tested were chosen consid- the type of analyzed matrix was calculated by dilution method
ering the highest dose used in standard methods to establish (Di Cesare et al. 2013) and no inhibition was obtained. The
resistance to antibiotics with clinical strains (breakpoint) ARG and intI1 abundances were expressed as absolute abun-
(CLSI 2018) or the concentration reported in previous dance (log gene copies/mL) and relative abundance (gene
study (Gao et al. 2012). Total heterotrophic count (HPC) copies/16S rRNA gene copy) and a mean value of the abun-
was determined on media without antibiotics. dance for each gene was calculated. The interpretation of the
Serial dilutions of wastewater samples (influent and efflu- results in case of abundance values lower than the LOQ and
ent) were plated in duplicate on media with and without anti- in case of discordance between the two replicates was made
biotics. Serial dilution of surface water or different volumes as previously reported (Di Cesare et al. 2015).

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Statistical analyses A generally higher ARB concentration and ARG relative

and absolute abundance were observed in the inlet of the
The statistical analysis of the data was carried out using treatment plants (influent of WWTPs and surface raw water
the SPSS package (Version 25.0) for Windows. The bacte- treated in the DWTPs) with respect to the outlet (effluent and
rial concentrations and ARG abundances were converted drinking water), although no differences were detected when
to log10 (log CFU/mL or log copies/mL), whereas relative considering the antibiotic resistance rates (%).
ARG abundances were transformed in the arcsine of their The sampling period seemed to not affect the dynamics
square root (arcsine of square root of gene copy/16 rRNA of antibiotic resistance.
gene copy) (Crawley 2012). For the comparison of ARB
concentration or antibiotic resistance rate or ARG abundance Dynamics of ARB and ARGs in WWTPs
between influent vs effluent in the WWTPs or raw surface
water vs drinking water in the DWTPs, the Student t-test was Total HPCs, ampicillin-resistant bacteria (AmRB), tetracy-
used. This test was also applied to evaluate the differences cline-resistant bacteria (TRB), and sulfonamide-resistant
between DWTP D and DWTP E. The one-way ANOVA test, bacteria (SRB) detected in the influents and in the effluents
followed by Tukey’s post hoc analysis, was used to study of the three WWTPs investigated are reported in Fig. 1. In
the differences among the sampling and the WWTPs. The the influents, the concentrations ranged from 5.7 to 6.4 for
relationship among ARGs (relative abundance) and between AmRB, from 4.5 to 6.1 for TRB, and from 5.4 to 7.0 log
ARGs (relative abundance) and ARB (antibiotic resistance CFU/mL for SRB. The highest mean concentration reported
rate) was analyzed with Pearson’s correlation test. was 6.4 log CFU/mL for SRB and the lowest 6.1 log CFU/
mL for AmRB. The mean resistance rates of AmRB, TRB,
and SRB were 9%, 1%, and 22%, respectively, and their
Results highest ratio reached up to 21%, 4%, and 38%.
The concentrations in the effluents ranged from 2.1 to
Dynamics of ARB and ARGs in urban water cycle 4.5 for AmRB, from 1.3 to 3.9 for TRB, and from 2.1 to
4.9 log CFU/mL for SRB. Moreover, the highest mean con-
The dynamics of antibiotic resistance in the urban water centration was 3.9 log CFU/mL for SRB and the lowest
cycle that includes WWTPs and DWTPs highlighted a statis- 2.8 log CFU/mL for TRB. As observed in the influent, the
tically significant difference (Table 1) of the ARB concentra- decreasing trend of the mean resistance rates of ARBs was
tions and ARGs (relative and absolute abundance) between SRB > AmRB > TRB (21% > 12% > 3%) and their highest
WWTPs and DWTPs, with higher values in the WWTPs ratio reached up to 35%, 33%, and 12%, respectively.
(Figs. 1, 2, 3, and 4). On the contrary, no difference was A statistically significant reduction of heterotrophic bac-
revealed for the antibiotic resistance rates (%) with respect teria and ARB was observed in WWTPs after the treatment
to the plant (WWTPs vs DWTPs). (Table 2, Fig. 1a). On the contrary, for all ARB monitored,

Table 1  Results of the Student t-test and the one-way ANOVA test considering the antibiotic-resistant bacteria (ARB) and antibiotic-resistant
genes (ARGs) in urban water cycle
Inleta vs ­outletb WWTPs vs DWTPs Sampling period

Concentration Antibiotic resistance Concentration Antibiotic resistance Concentration Antibiotic resistance

rate rate rate
HPC p < 0.001 n.s p < 0.05 n.s n.s n.s
AmRB p < 0.001 n.s p < 0.05 n.s n.s n.s
TRB p < 0.001 n.s p < 0.05 n.s n.s n.s
SRB p < 0.001 n.s p < 0.05 n.s n.s n.s
Absolute abundance Relative abundance Absolute abundance Relative abundance Absolute abundance Relative abundance
blaTEM p < 0.001 p < 0.001 p < 0.001 p < 0.001 n.s n.s
tetA p < 0.001 p < 0.001 p < 0.001 p < 0.001 n.s n.s
sulII p < 0.001 p < 0.001 p < 0.001 p < 0.001 n.s n.s
intI1 p < 0.05 p < 0.05 n.s p < 0.001 n.s n.s

HPC, total heterotrophic count; AmRB, ampicillin-resistant bacteria; TRB, tetracycline-resistant bacteria; SRB, sulfonamide-resistant bacteria;
n.s., not significant
a
Influents of WWTPs + drinking water source
b
Effluents of WWTPs + drinking water

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Fig. 1  a Log-transformed

concentrations of ampicillin-,
tetracycline-, sulfonamide-
resistant bacteria and HPC
in influent and effluent of the
WWTPs investigated. Box plots
represent median and range
values. b Antibiotic resistance
rate of ampicillin-, tetracycline-,
and sulfonamide-resistant bac-
teria in WWTPs. All values are
normalized to HPC abundances.
Box plots represent median
and range values. WWTPs,
wastewater treatment plants;
HPC, total heterotrophic count;
AmRB, ampicillin-resistant bac-
teria; TRB, tetracycline-resist-
ant bacteria; SRB, sulfonamide-
resistant bacteria

the antibiotic resistance rate did not show a significant trend respectively (Fig. 2a). The relative abundances of blaTEM,
(influent vs effluent, Table 2), except for TRB (higher value tetA, sulII, and intI1 with respect to 16S rRNA gene, when
in the effluent of WWTPs) (Fig. 1b). Moreover, no difference quantifiable, ranged from 3.3 × ­10−4 to 9.1 × ­10−4, 1.1 × ­10−3
was observed among the different WWTPs both considering to 3.7 × ­10−3, 2.0 × ­10−1 to 8.7 × ­10−1, and 1.6 × ­10−3 to
the results expressed as log CFU/mL and as antibiotic resist- 3.7 × ­10−3 copies/16S rRNA gene copy, respectively. sulII
ance rates. Also the sampling period seemed not to affect the showed for both absolute and relative abundances the
abundances and the rates of TRB, SRB, and AmRB. highest mean values (mean: 9.4 log gene copies/mL and
The absolute abundance of bla TEM, tetA, sulII, and 3.8 × ­10−1 copies/16S rRNA gene copy) (Fig. 2).
intI1 in influents, when quantifiable, ranged from 8.0 to The absolute abundance of bla TEM, tetA, sulII, and
9.0, 8.7 to 9.4, 8.8 to 9.8, and 8.4 to 9.5 log gene copies/mL, intI1 when quantifiable ranged in the effluents from 7.3 to

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Fig. 2  a Absolute abundance

of tetA, sulII, blaTEM, and intI1
gene in influent and effluent
of the WWTPs investigated.
Box plots represent median and
range values. b Relative abun-
dance of tetA, blaTEM, intI1 (b1)
and sulII (b2) gene in WWTPs.
All values are normalized to
16S rRNA gene copy. Box plots
represent median and range
values. WWTPs, wastewater
treatment plants

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Fig. 3  a Log-transformed

concentrations of ampicillin-,
tetracycline-, sulfonamide-
resistant bacteria, and HPC
in surface and drinking water
of the DWTPs investigated.
Box plots represent median
and range values. b Antibiotic
resistance rate of ampicillin-,
tetracycline-, and sulfonamide-
resistant bacteria in DWTPs.
All values are normalized to
HPC abundances. Box plots
represent median and range
values. DWTPs, drinking water
treatment plants; HPC, total
heterotrophic count; AmRB,
ampicillin-resistant bacteria;
TRB, tetracycline-resistant
bacteria; SRB, sulfonamide-
resistant bacteria

8.2, 7.6 to 9.2, 8.8 to 10.3, and 8.4 to 9.6 log gene copies/ Although the absolute abundance of ARGs generally did
mL, respectively (Fig. 2a). The relative abundances when not show differences between the influents and effluents of
quantifiable of bla TEM was 1.9 × ­10−4 copies/16S rRNA WWTPs, a significant reduction of the relative abundance of
gene copy, and the relative abundance of tetA, sulII, and tetA, blaTEM, and intI1 was observed in the effluents.
intI1 ranged from 2.7 × ­1 0 −5 to 1.5 × ­1 0 −3, 1.3 × ­1 0 −1 to Significant differences among the WWTPs were
9.1 × ­10−1, and 5.6 × ­10−4 to 3.1 × ­10−3 copies/16S rRNA reported in effluents only for the relative abundance
gene copy. As observed already in the influents, the high- of sulII (WWTP2 > WWTP1 and WWTP3), tetA
est mean value was obtained for sulII (mean: 9.5 log gene (WWTP1 > WWTP2), and intI1 (WWTP1 > WWTP2).
copies/mL and 3.6 × ­1 0 −1 copies/16S rRNA gene copy) The absolute and relative abundances were not influ-
(Fig. 2). enced by the sampling period.

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Fig. 4  a Absolute abundance

of tetA, sulII, blaTEM, and intI1
gene in surface and drinking
water of the DWTPs inves-
tigated. Box plots represent
median and range values. b
Relative abundance of tetA,
blaTEM, sulII, and intI1 gene in
DWTPs. All values are normal-
ized to 16S rRNA gene copy.
Box plots represent median and
range values. DWTPs, drinking
water treatment plants

A correlation between relative abundance of intI1 vs tetA ARB were observed in all analyzed surface water sam-
(p < 0.001), intI1 vs blaTEM (p < 0.05), and blaTEM vs tetA ples with concentrations ranging between 4.7 and 6.3 for
(p < 0.001) was observed (p < 0.05). No relationship was AmRB, 3.3 and 6.2 for TRB, and 4.9 and 6.9 log CFU/mL
reported for ARGs and ARB, except a correlation between for SRB (Fig. 3a). Moreover, the highest mean concentra-
TRB and tetA (p < 0.05) and TRB and intI1 (p < 0.05). tion was 6.1 log CFU/mL for SRB and the lowest 4.9 log
CFU/mL for TRB. The highest ratio is 36% for SRB and
Dynamics of ARB and ARGs in DWTPs the lowest 5% for TRB.
Considering the drinking water, SRB were observed in
ARB concentration and antibiotic resistance rate (%) all analyzed samples with concentrations ranging from 1.0
detected in surface and drinking water of the two DWTPs to 2.1 log CFU/mL. On the contrary, TRB and AmRB were
are reported in Fig. 3. found in the 50% and 25% of the analyzed samples and the

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Table 2  Results of the Student t-test and the one-way ANOVA test considering the antibiotic-resistant bacteria (ARB) and antibiotic-resistant
genes (ARGs) in wastewater treatment plants (WWTPs)
Influent vs effluent Wastewater treatment Sampling period

Concentration Antibiotic resistance Concentration Antibiotic resistance Concentration Antibiotic resistance

rate rate rate
HPC p < 0.001 n.s n.s n.s n.s n.s
AmRB p < 0.001 n.s n.s n.s n.s n.s
TRB p < 0.001 p < 0.05 n.s n.s n.s n.s
SRB p < 0.001 n.s n.s n.s n.s n.s
Absolute abundance Relative abundance Absolute abundance Relative abundance Absolute abundance Relative abundance
blaTEM n.s p < 0.001 n.s n.s n.s n.s
tetA n.s p < 0.001 n.s p < 0.05a n.s n.s
sulII n.s n.s n.s p < 0.05a n.s n.s
intI1 n.s p < 0.001 n.s p < 0.05a n.s n.s

HPC, total heterotrophic count; AmRB, ampicillin-resistant bacteria; TRB, tetracycline-resistant bacteria; SRB, sulfonamide-resistant bacteria;
n.s., not significant
a
Only for the effluent

concentration when quantifiable ranged from 1.2 to 1.9 log copies/mL, respectively (Fig. 4a). The relative abundances
CFU/mL for AmRB and from 0.4 to 1.5 log CFU/mL for of tetA, sulII, and intI1 ranged from 5.9 × ­10−5 to 2.6 × ­10−4,
TRB. Moreover, the highest mean concentration was 1.5 5.8 × ­1 0 −5 to 9.1 × ­1 0 −4, and 1.8 × ­1 0 −4 to 1.5 × ­1 0 −3
log CFU/mL for SRB and the lowest 0.4 log CFU/mL for copies/16S rRNA gene copy, respectively. intI1 showed
AmRB. According to the ARB concentration, the highest the highest mean values for both absolute and relative
resistance rate was observed for SRB (mean value: 27%). abundances (mean: 9.6 log gene copies/mL and 7.5 × ­10−4
A significant reduction (Table 3) was detected after the treat- copies/16S rRNA gene copy) (Fig. 4b).
ments for both heterotrophic bacteria and ARB (log CFU/mL) blaTEM and tetA were not detected in drinking waters,
in DWTP (Fig. 3a), while no abatement was observed for anti- except for three samples (1 for DWTP D, 2 for DWTP E)
biotic resistance rates (Fig. 3b). No difference was observed that presented only one replicate positive but not quantifiable
between the DWTPs and sampling period did not impact on for tetA, while all drinking water samples were positive for
abundances and rates of TRB, SRB, and AmRB. sulII, although this gene was not quantifiable. The absolute
In all the surface water sample, intI1, tetA, and sulII and relative abundance of intI1 was 7.5 log gene copies/mL
were observed, while blaTEM was never detected. The abso- and 1.3 × ­10−3 copies/16S rRNA gene copy, respectively.
lute abundance of tetA, sulII, and intI1 in these samples The relative and absolute abundance of tetA and sulII
ranged from 8.2 to 9.6, 8.6 to 10.0, and 8.4 to 10.3 log gene were reduced by each water treatment; moreover, for DWTP

Table 3  Results of the Student t-test and the one-way ANOVA test considering the antibiotic-resistant bacteria (ARB) and antibiotic-resistant
genes (ARGs) in drinking water treatment plants (DWTPs)
Surface water vs drinking water Water treatment Sampling period

Concentration Antibiotic resistance Concentration Antibiotic resistance Concentration Antibiotic resistance

rate rate rate
HPC p < 0.001 n.s n.s n.s n.s n.s
AmRB p < 0.001 n.s n.s n.s n.s n.s
TRB p < 0.001 n.s n.s n.s n.s n.s
SRB p < 0.001 n.s n.s n.s n.s n.s
Absolute abundance Relative abundance Absolute abundance Relative abundance Absolute abundance Relative abundance
blaTEM n.d n.d n.d n.d n.d n.d
tetA p < 0.001 p < 0.001 n.s n.s n.s n.s
sulII p < 0.001 p < 0.001 n.s n.s n.s n.s
intI1 p < 0.05 n.s n.s p < 0.05 n.s n.s

HPC, total heterotrophic count; AmRB, ampicillin-resistant bacteria; TRB, tetracycline-resistant bacteria; SRB, sulfonamide-resistant bacteria;
n.s., not significant; n.d., not determined

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E also, the absolute abundance of intI1 was lower in the Although the treatment process, the dimension, and the
drinking water (Fig. 4a). The relative abundance of intI1 in quality of the treated effluent varied in the investigated
the drinking water was the sole parameter evidencing a dif- WWTPs, no significant difference in presence and abun-
ference between the DWTPs (DWTP E > DWTP D; Fig. 4b). dance of ARB and ARGs was observed among the three
The absolute and relative ARG abundances were not influ- WWTPs, highlighting that these factors had a limited impact
enced by the sampling period (Table 3). on the fate of the resistances released by the plants. This is in
Moreover, both relative and absolute abundances of tetA agreement with the literature that hypothesizes a role for the
showed a correlation with the absolute abundance of sulII biological process, present in all the investigated WWTPs, in
(p < 0.001). No relationship was detected between the values the establishment of a bacterial community exerting specific
of ARGs and ARB, except for a correlation between SRB ecological factors (e.g., enhanced competition, predation,
and sulII gene (p < 0.001). cooperation) determining the presence of ARB and ARGs
(Rizzo et al. 2013; Manaia et al. 2016).
As previously observed among ARB and ARGs investi-
Discussion gated, SRB and sulII were the most abundant (Munir et al.
2011; Ferro et al. 2016; Ben et al. 2017).
The urban water cycle that comprises both wastewater and In order to evaluate the dynamics of antibiotic resistance
drinking water treatment represents a possible route for in the urban water cycle, it is also important to consider
spreading of ARB and ARGs into the environment with a the spread of ARB and ARGs in surface waters utilized for
subsequently potential return to humans with a direct impact drinking purposes that are influenced by the discharge of
on the human health (Manaia 2017). The treatment plants wastewater and by other anthropogenic activities. The role
(WWTPs and DWTPs) investigated in our study, being in of surface water is confirmed in our study by the presence of
the same geographical area, are particularly interesting to all ARB and most of the quantified genes in all samples of
track the fate of ARB and ARGs in the urban water cycle. raw surface water analyzed used as drinking water source,
The overall ARB and ARG dynamics in the WWTPs and with high abundances of SRB and sulII, according to the
DWTPs sampled in this study highlighted, as expected, higher results obtained in the three WWTPs that are in the same
concentrations of antibiotic resistances in the WWTPs than in geographical area. The high abundance of antibiotic resist-
DWTPs. This is in agreement with several studies that identi- ances against sulfonamides in surface water was observed
fied the WWTP as hotspot for the spread of antibiotic resist- also in other rivers impacted by human activities (Hu et al.
ances (Rizzo et al. 2013; Pazda et al. 2019). The concentra- 2019; Yang et al. 2020). The results obtained in the inves-
tions of ARB in the influent of WWTPs observed in our study tigated surface water revealed a higher absolute abundance
were similar or higher than those reported in other studies of ARGs and ARB (3–4 orders of magnitude) with respect
for TRB and SRB (Munir et al. 2011; Gao et al. 2012) and, to the data reported for other rivers in China (Hu et al. 2019;
generally, the monitored ARGs showed absolute abundances Yang et al. 2020) and Poland (Bondarczuk and Piotrowska-
substantially higher than those reported by other authors Seget 2019) influenced by anthropogenic activities (e.g.,
(Narciso-da-Rocha et al. 2018; Fiorentino et al. 2019; Wang sewage discharges, agricultural runoff, swine farm) under-
et al. 2020). The reduction of ARB concentrations between lining its role in the spreading of antibiotic resistance. The
the influent and the effluent of the studied WWTPs was com- presence of ARB and ARGs in surface water used as drink-
parable with similar studies (Wang et al. 2020). ing water source highlights the importance to investigate all
Moreover, the stability of the antibiotic-resistant rates in the phases composing the urban water cycle and to deepen
the influents and effluents (proportion of ARB in the HPC the effect of the drinking water treatment.
for each sample) underlined that the different treatments did None of the detected ARB or ARGs was completely
not promote the selection of ARB. This was also confirmed removed in drinking water by the applied water treatments,
by the relative abundance of the ARGs that showed a reduc- according to Hu et al. (2019) and Siedlecka et al. (2021)
tion for blaTEM, tetA, and intI1, with values significantly who investigated ARGs in a DWTP in China and Poland.
lower in the effluent. The results reported in literature that As previously reported in other studies, the presence and
considered the effect of the treatment on the variation of the total concentration of ARGs in drinking water signifi-
the ARG relative abundances are discordant. Indeed, in cantly decreased in comparison to corresponding water
two studies, performed in Canada and in China, no signifi- source (Sanganyado and Gwenzi 2019; Stange et al. 2019).
cant differences were observed in the relative abundance of Moreover, the limited variations in antibiotic resistance
ARGs between influent and effluent of two WWTPs (Mao rates between surface water and drinking water associated
et al. 2015; McConnell et al. 2018). On the contrary, Di with the reduction of relative ARG abundances showed,
Cesare et al. (2016) showed an increase of some ARGs (e.g., as observed in WWTPs, that the treatment did not apply
sulII) and of intI1 in the effluents of three Italian WWTPs. any selective pressure. These results highlight the key role

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played by the drinking water treatment in reducing ARGs influenced by the physiological status of bacteria; these
and controlling ARB from the water source. As observed for characteristics make this method a useful tool to evaluate
WWTPs, the different drinking water treatments seemed to the potential of antibiotic resistance spreading in environ-
display a limited impact on the spread of SRB and ARGs, ment (Rizzo et al. 2013). However, as highlighted before,
although DWTP D was equipped with an ozonation step qPCR is a target-based quantification method that cannot
that resulted the best treatment to reduce ARB and ARGs provide a complete overview of the antibiotic resistome of
in some studies (Stange et al. 2019; Yang et al. 2020). It is a microbial community (Hendriksen et al. 2019).
important to highlight that the effectiveness of the disinfec-
tion step can be influenced by numerous factors such as the
bacterial community composition in the raw water used as
drinking water source, the effectiveness of other treatment Conclusion
steps, and the co-selection of antibiotic resistance enhanced
by disinfection by-products (Sanganyado and Gwenzi, The results obtained underline that to investigate the antibiotic
2019). resistance dynamics in the same geographical area is particu-
Despite the observed reduction after drinking water treat- larly interesting to track the fate of ARB and ARGs in the
ment, the presence of ARB and ARGs in drinking water urban water cycle. More detailed description of the phenom-
should be kept under observation, considering that ARB enon was obtained in this study integrating the information
may also enter the human microbiome following water con- obtained by culture-dependent method with those obtained
sumption (Vaz-Moreira et al. 2014) and ARGs can occasion- by the culture-independent one. However, the results claim
ally be transferred to human pathogenic bacteria (Manaia for the need to perform more studies by using untargeted
2017). Moreover, it should be taken into account that the based quantification approach, i.e., shotgun metagenomics,
amount of ARB could increase in tap water, due to biofilm to characterize the overall antibiotic resistome (total content of
detachment in the distribution system (Zhang et al. 2018). ARGs) of a microbial community aiming to find the best tar-
Considering that some genes can be utilized as proxy for the gets as proxy of the antibiotic resistome in aquatic ecosystems.
overall abundance of ARGs in waters (Su et al. 2018), Pear- Although the processes applied for treating wastewater
son’s correlation was carried out among the different measured and drinking water allowed to reduce the concentration of
ARGs in WWTPs and DWTPs to evaluate the suitability of ARB and ARGs and did not apply selective pressures, the
the monitored target as indicator. The results highlighted that results highlighted the importance to monitor the presence
a positive correlation was observed only for blaTEM vs tetA in of ARB and ARGs mainly in drinking water that repre-
WWTPs and sulII vs tetA in DWTPs underlining that none sents a potential route of transmission to humans with a
of the selected genes could be used to predict the fate and the direct impact on human health.
potential contamination by determinants of antibiotic resistance
Supplementary Information The online version contains supplemen-
in the urban water cycle. A similar result was also observed for tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11356-0​ 22-2​ 4650-w.
intI1 that was correlated only with blaTEM and tetA in WWTPs.
Another important topic is related to the meaning of Author contribution All authors contributed to the study conception
the results obtained with the two approaches (cultiva- and design. Experiments were performed by CP, MP, MM, and RS,
while data collection and analysis was done by SiB, SaB, AD, and
tion dependent and independent) utilized in this study RS. The first draft of the manuscript was written by SiB, SaB, and AD
for characterizing the antibiotic resistance. Molecular and all authors commented on previous versions of the manuscript.
results highlighted a trend (influent > effluent; raw surface GC and EC were in charge of supervision and greatly contributed to
water > drinking water) that was not shown with cultiva- final review and editing of the paper. All authors read and approved
the final manuscript.
tion-dependent methods; moreover, no clear relationship
between ARB and the corresponding ARGs was observed. Data availability All data generated or analyzed during this study are
This is probably related to the different information that included in this published article.
the two approaches provide. The cultivation-dependent
method offers a general overview of resistant bacteria Declarations
but considers only a part of the viable bacteria because Ethics approval This is an original article that did not use other infor-
no information about VBNC (viable but not culturable mation that requires ethical approval.
bacteria) can be provided. Moreover, other drawbacks
associated with this approach are present; for example, Consent to participate No consent of participation is to be claimed.
the results obtained can be influenced by the antibiotic Consent for publication All of the authors have read and approved the
tested and the concentration used (Manaia et al. 2016). paper for publication. We confirmed that it has not been published pre-
On the contrary, the cultivation-independent method has viously nor is it being considered by any other peer-reviewed journal.
high specificity and sensitivity and the detection is not

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Environmental Science and Pollution Research (2023) 30:35294–35306 35305

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scito​tenv.​2018.​11.​211

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