Prevalence of ESBL-producing E. coli in Dutch recreational waters

Prevalence and characteristics of ESBL-producing E. coli in
Dutch recreational waters influenced by wastewater
treatment plants
Hetty Blaak a,
*, Patrick de Kruijf a
, Raditijo A. Hamidjaja a
,
Angela H.A.M. van Hoek a
, Ana Maria de Roda Husman a,b
,
Franciska M. Schets a
a
National Institute for Public Health and the Environment (RIVM), Centre for Zoonoses and Environmental Microbiology, PO Box 1,
3720 BA Bilthoven, The Netherlands
b
Institute for Risk Assessment Sciences, Utrecht University, PO Box 80178, 3508 TD Utrecht, The Netherlands
1. Introduction
During the last two decennia, the prevalence of Extended
Spectrum Beta-Lactamase (ESBL)-producing bacteria has
increased worldwide (Cantoń et al., 2008; Castanheira et al.,
2008). ESBL-producing bacteria are resistant to most beta-
lactam antibiotics, including 3rd and 4th generation
cephalosporins, and are often additionally resistant to
multiple other classes of antibiotics. This severely limits
treatment options for infections caused by these bacteria,
which has led to an increased use of last-resort antibiotics
such as carbapenems (Cantoń et al., 2012).Although initially
Veterinary Microbiology 171 (2014) 448–459
A R T I C L E I N F O
Keywords:
ESBL
E. coli
Recreational water
Wastewater treatment plants
A B S T R A C T
Outside health care settings, people may acquire ESBL-producing bacteria through
different exposure routes, including contact with human or animal carriers or
consumption of contaminated food. However, contact with faecally contaminated surface
water may also represent a possible exposure route. The current study investigated the
prevalence and characteristics of ESBL-producing Escherichia coli in four Dutch
recreational waters and the possible role of nearby waste water treatment plants
(WWTP) as contamination source. Isolates from recreational waters were compared with
isolates from WWTP effluents, from surface water upstream of the WWTPs, at WWTP
discharge points, and in connecting water bodies not influenced by the studied WWTPs.
ESBL-producing E. coli were detected in all four recreational waters, with an average
concentration of 1.3 colony forming units/100 ml, and in 62% of all samples. In surface
waters not influenced by the studied WWTPs, ESBL-producing E. coli were detected in
similar concentrations, indicating the existence of additional ESBL-E. coli contamination
sources. Isolates with identical ESBL-genes, phylogenetic background, antibiotic resistance
profiles, and sequence type, were obtained from effluent and different surface water sites
in the same watershed, on the same day; occasionally this included isolates from
recreational waters.
Recreational waters were identified as a potential exposure source of ESBL-producing E.
coli. WWTPs were shown to contribute to the presence of these bacteria in surface waters,
but other (yet unidentified) sources likely co-contribute.
ß 2014 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +31 30 274 7005; fax: +31 30 274 4434.
E-mail address: hetty.blaak@rivm.nl (H. Blaak).
Contents lists available at ScienceDirect
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
http://dx.doi.org/10.1016/j.vetmic.2014.03.007
0378-1135/ß 2014 Elsevier B.V. All rights reserved.

ESBL-production was typically associated with hospital-
acquired infections caused by Klebsiella pneumoniae, it is
now also associated with community-acquired infections,
mainly urinary tract infections caused by Escherichia coli
(Livermore et al., 2007; Paterson and Bonomo, 2005).
Moreover, ESBL-producing E. coli are present among the
commensal E. coli population in healthy individuals and
food-producing animals (Huijbers et al., 2013; Nethmap_-
MARAN, 2013; Trott, 2013). Commensal E. coli generally do
not cause disease, however, spread of ESBL-producing
variants through human and animal populations is never-
theless worrisome, and may lead to increased exposure of
populations more susceptible to opportunistic infections
(e.g. the elderly or hospitalized individuals). Additionally,
with an increasing number of ESBL-producing E. coli carriers
in the human population the risk increases that gut
pathogens efficiently acquire resistance by gene transfer
of ESBL-genes as well as other antibiotic resistance genes
from ESBL-producing E. coli in the intestinal tract.
Dissemination of ESBL-producing E. coli outside the
healthcaresettingmaybefacilitatedbycontact withhuman
or animal carriers, or consumption of contaminated animal
products. Additionally, a possible role for the environment
should be considered in this regard. Since E. coli are
commensal bacteria, they are abundantly excreted into
the environment, amongst others through application of
manure as fertilizer or droppings of pasture animals, with
feces of wild animals, and with discharge of (partially)
treated wastewater, or with sewage overflows during heavy
rainfall. Some of the commensal as well as pathogenic E. coli
that are excreted into the environment may have the
capacity to produce ESBL. Indeed, ESBL-producing E. coli
have been detected in surface water worldwide, including
the Netherlands (Blaak et al., 2011; Chen et al., 2010; Dhanji
et al., 2011; Hong et al., 2004). Human exposure to these
bacteria may occur, for instance during recreation in
contaminated surface water, or indirectly, when contami-
nated surface water is used for irrigation of (raw consumed)
crops, therewith contributing to community-associated
dissemination of ESBL-producing E. coli. Additionally,
contaminated surface water might contribute to exposure
of animals (wild life as well as livestock) that drink from it.
In order to limit dissemination of ESBL-producing E. coli
through the environment, insight in the contribution of
different possible environmental contamination sources
and exposure routes is essential. The current study
determined the presence of ESBL-producing E. coli in four
Dutch recreational water regions, and the possible
contribution of nearby wastewater treatment plants.
2. Materials and methods
2.1. Sampling and sampling locations
Three recreational waters appointed under European
Bathing Water Directive 2006/7/EC (‘official’) (Anony-
mous, 2006) and one not appointed (‘unofficial’) recrea-
tional water were sampled during the bathing seasons of
2011 and/or 2012 which, in the Netherlands, lasts from
May 1st until September 30th. The recreational waters
were situated in different regions in the Netherlands, and
each was located 1–2 km (as the crow flies) from a
wastewater treatment plant (WWTP) that did not disinfect
treated effluents before discharge (Fig. 1). Two of the
official recreational waters (regions A and B) were situated
in freshwater lakes; the third (region C) was located in the
Fig. 1. Sampling locations in regions A, B, C, and D. *For regions A and B, the direction of currents is variable and ‘upstream’ and ‘downstream’ annotations are
based on the main current direction. ** The entire river is used for recreation, the indicated site is chosen to represent recreational water downstream of an
WWTP.
H. Blaak et al. / Veterinary Microbiology 171 (2014) 448–459 449

North Sea. The fourth and unofficial recreational water
(region D) was a small river that is frequently used for
canoeing. All recreational waters were connected to the
surface water where WWTP effluents were discharged
(‘discharge points’). At both lake-side recreational waters
in regions A and B, the direction of the current is variable,
depending on wind direction and operational schedules of
water pumping engines and/or sluices. In both cases the
main current direction is from WWTP discharge points to
recreational waters, and for descriptional simplicity, these
recreational waters are referred to as located ‘downstream’
of the WWTPs. The currents at WWTP discharge points in
region C and D are always directed from east to west. The
small river in region D was sampled both upstream and
downstream of the WWTP situated at the river bank, of
which the downstream sample was taken to represent
recreational water under influence of the WWTP.
In 2011, recreational waters in regions A–C were
sampled four or five times during the bathing season.
During the 2012 bathing season, waters were sampled
three (A, C, D) or four (B) times (Table 1), and at each
sampling time-point additional samples were taken from
WWTP effluents, surface water at WWTP discharge points,
surface water located upstream of the WWTPs, and surface
waters from connecting water bodies upstream of the
recreational waters but not under influence of the studied
WWTPs (Fig. 1). In region B, surface water ‘upstream of the
WWTP’ was not sampled. Instead, because of the presence
of two dams between WWTP and the recreational water
(possibly interfering with passage of water between the
two sites), an additional surface water sample ‘down-
stream’ of the WWTP, but ‘upstream’ of the recreational
water was taken. Due to logistic problems, discharge
points were only sampled twice in regions B and D.
Prior to the day of sampling of surface water, effluents
were collected by WWTP staff using automated systems
that continuously sample effluent, rendering 24 h flow-
proportional, homogenous samples that are collected in
mixing vessels. One liter of effluent sample was taken from
these mixing vessels. On the third sampling date for region
C, no 24 h flow-proportional effluent sample was available,
and a 1 l grab sample was taken from the effluent duct on
the morning of surface water sampling. Surface water
samples (1 l) were taken according to NEN-EN-ISO 19458
(Anonymous, 2007). Samples were transported and stored
at 5 Æ 3 8C, and analyzed within 24 h after sampling.
2.2. Isolation and enumeration of E. coli and ESBL-producing
E. coli
From each sample, multiple volumes were filtered
through 0.45 mm pore size membrane filters (Millipore,
Amsterdam, the Netherlands), and E. coli was isolated
according to ISO 9308-1 ‘Rapid test’ (Anonymous, 2000). In
short, filters were incubated on TSA for 4–5 h at 36 Æ 2 8C,
and then transferred to TBA and incubated for 19–20 h at
44 Æ 0.5 8C. Presumptive E. coli colonies were stained with
James reagent to test for indole production (Biomerieux,
Table 1
Concentrations ESBL-E. coli and E. coli in Dutch recreational waters.
Region Sampling date Concentrations (cfu/100 ml) (95%-Confidence Interval) % ESBL
ESBL-E. coli E. coli
A 4-7-2011 0.29 (0.05–0.88) 2.9 Â 102
(2.2 Â 102
–3.6 Â 102
) 0.10
25-7-2011 15 (12–18) 1.8 Â 103
(1.6 Â 103
–1.9 Â 103
) 0.83
8-8-2011 1.9 (1.1–3.1) 1.2 Â 103
(7.4 Â 102
–1.7 Â 103
) 0.17
6-9-2011 0.15 (0.0084–0.65) 67 (52–84) 0.22
18-6-2012 1.0 (0.44–2.0) 4.8 Â 102
(4.0 Â 102
–5.8 Â 102
) 0.21
2-7-2012 1.2 (0.54–2.2) 1.3 Â 102
(1.1 Â 102
–1.5 Â 102
) 0.93
13-8-2012 0 (0–0.33)a
83 (69–99) <0.4
B 7-6-2011 4.8 (3.7–6.1) 5.1 Â 103
(4.2 Â 103
–6.2 Â 103
) 0.093
4-7-2011 0 (0.00–0.27) 1
1.4 Â 102
(1.1 Â 102
–1.6 Â 102
) < 0.2
25-7-2011 0.15 (0.0084–0.65) 2.7 Â 102
(2.1 Â 102
–3.4 Â 102
) 0.055
8-8-2011 0 (0.00–0.28) 1
2.3 Â 102
(2.0 Â 102
–2.6 Â 102
) <0.1
6-9-2011 0.15 (0.0084–0.65) 2.1 Â 102
(1.6 Â 102
–2.8 Â 102
) 0.069
21-5-2012 0 (0–0.28)a
23 (15.0–33.1) <1.2
2-7-2012 0 (0–0.28)a
32 (24.7–40.2) <0.9
13-8-2012 0 (0–0.28)a
48 (39.6–57.0) <0.6
24-9-2012 0.29 (0.049–0.91) 92 (75.5–111.6) 0.32
C 21-6-2011 0 (0.00–0.64)a
31 (21.3–43.2) <2
4-7-2011 0 (0.00–0.27)a
29 (19.7–40.9) <0.9
25-7-2011 3.4 (2.2–5.0) 6.2 Â 102
(5.2 Â 102
–7.4 Â 102
) 0.54
8-8-2011 0.17 (0.01–0.76) 34 (23.8–46.7) 0.51
6-9-2011 1.0 (0.44–2.0) 1.3 Â 102
(1.1 Â 102
–1.6 Â 102
) 0.77
4-6-2012 0 (0–0.29)a
23 (14.5–33.1) <1.3
16-7-2012 6.2 (4.5–8.2) 7.5 Â 102
(6.1 Â 102
–9.2 Â 102
) 0.82
27-8-2012 0.29 (0.049–0.91) 45 (22.8–79.7) 0.65
D 18-6-2012 0.29 (0.049–0.91) 3.1 Â 102
(2.8 Â 102
–3.4 Â 102
) 0.10
30-7-2012 8.1 (6.1–10) 8.3 Â 102
(7.2 Â 102
–9.6 Â 102
) 0.97
10-9-2012 0.15 (0.0084–0.65) 2.6 Â 102
(2.3 Â 102
–2.8 Â 102
) 0.057
a
No ESBL-producing E. coli were detected. cfu = colony forming units.
H. Blaak et al. / Veterinary Microbiology 171 (2014) 448–459450

Boxtel, the Netherlands), and only indole-positive colonies
were counted. Indole positive isolates were further confirmed
by testing for ß-glucuronidase-activity on Brilliance E. coli/
coliform agar (BECSA; Oxoid, Badhoevedorp, the Netherlands).
Beta-glucuronidase-positive, indole-positive isolates were
considered to be E. coli. API20E (Biomerieux) was used to
determine whether or not indole-positive, ß-glucuronidase-
negative isolates were E. coli. E. coli concentrations were
calculated based on the number of indol-positive colonies and
the fraction of these colonies that was confirmed to be E. coli.
For the isolation and enumeration of ESBL-producing E.
coli, membrane filters were placed on ChromIDTM
ESBL
agar (Biomerieux) and incubated for 18–24 h at 36 Æ 2 8C.
Species identity of suspected ESBL-E. coli isolates (i.e. ß-
glucuronidase-positive on ChromIDTM
ESBL agar) were
confirmed by indole-testing using BBL Dry SlideTM
(BD,
Breda, The Netherlands). Suspected ESBL-producing isolates
were tested for ESBL-production by disk diffusion following
CLSI guidelines (Clinical and Laboratory Standards Institute,
2010), using Sensi-DiscTM
(BD, Breda, the Netherlands)
according to the manufacturer’s instructions. Zone diameters
were determined for cefotaxime (30 mg/ml), cefotaxime
(30 mg/ml) + clavulanic acid (10 mg/ml), ceftazidime (30ug/
ml), ceftazidime (30ug/ml) + clavulanic acid (10ug/ml) and
cefoxitin (30 mg/ml). ESBL-producing isolates were defined
as strains resistant to cefotaxime (zone diameter 22 mm)
and/or ceftazidime (zone diameter 17 mm), and a reduction
in zone diameter of !5 mm with the disks containing
clavulanic acid (Clinical and Laboratory Standards Institute,
2010). Isolates without a significant effect of clavulanic acid
and resistant to cefoxitin (zone diameter 14 mm) were
considered AmpC-producing (Jacoby, 2009). Because of the
low isolation frequency of AmpC-producing isolates (8 of
394), these were excluded from further analyses. E. coli and
ESBL-producing E. coli concentrations and 95%-confidence
intervals (CI) were calculated using Mathematica software
9.0.1 (WolframResearch, Champaign, IL, USA).
2.2.1. Antibiotic resistance profiles
Using Sensi-DiscsTM
, phenotypically confirmed ESBL-
producing E. coli isolates (n = 386) were tested for
susceptibility to 12 antibiotics of human and veterinary
clinical relevance: tetracycline, ampicillin, amoxicillin/
clavulanic acid (co-amoxiclav), gentamicin, streptomycin,
sulfisoxazole, trimethoprim, chloramphenicol, ciprofloxa-
cin, nalidixic acid, imipenem, and meropenem. Tests were
performed according to CLSI guidelines following the
manufacturer’s instructions, and resistance determined
using CLSI breakpoints (Clinical and Laboratory Standards
Institute, 2010).
2.3. Phylogenetic typing
Of 386 confirmed ESBL-producing E. coli isolates with
known antibiotic resistance profiles, 214 were selected for
phylogenetic group analysis. Of these, 123 were randomly
selected across samples and from isolates with different
ABR phenotypes (defined as ‘random subset’), and 91 were
selected because of an observed match in phenotype with
respect to ABR profile between isolates from different
sampling sites within the same region at the same
sampling time-point (defined as ‘matches subset). Isolates
were allotted to phylogenetic groups A, B1, B2 or D, using a
PCR targeted to the chuA, yjaA genes and TspE4.C2 DNA
fragment, using primers described by Clermont et al.
(2000). Material from one single colony was suspended in
Tris EDTA buffer (pH 8.0, Sigma-Aldrich, Zwijndrecht, the
Netherlands) and cells were lysed at 70 8C for 5 min. DNA-
extracts were stored at À20 8C. Targets were amplified
using QIAGEN Multiplex PCR kit (Qiagen Benelux BV,
Venlo, the Netherlands), in 1.5 ml of 10Â diluted DNA
extract and using 5 pmol of each primer. Amplification
conditions were adapted from Clermont et al. (2000):
15 min 95 8C, followed by 35 cycles of 10 s 95 8C, 20 s 60 8C,
30 s 72 8C, and a final elongation step of 5 min 72 8C. Strains
were sub-grouped according to (Escobar-Paramo et al.,
2006): subgroup A0: chuAÀ, yjaAÀ, and TspE4.C2À;
subgroup A1: chuAÀ, yjaA+, and TspE4.C2À; group B1:
chuAÀ, yjaAÆ, and TspE4.C2+; subgroup B22: chuA+, yjaA+,
and TspE4.C2À; subgroup B23: chuA+, yjaA+, and TspE4.C2+;
subgroup D1: chuA+, yjaAÀ, and TspE4.C2À; subgroup D2:
chuA+, yjaAÀ, and TspE4.C2+.
2.4. ESBL genotyping
ESBL-genotypes were determined for a subset of 154 of
214 isolates with determined phylogroup, including 70
from the ‘random subset’ and 56 from the ‘matches subset’
(which were identified as matches with respect to
phylogroup as well as ABR profiles). Additionally, ESBL-
genotypes were determined for 28 isolates with unique
ABR profiles that were not included in phylogenetic group
analysis. Isolates were analyzed for the presence of genes
encoding CTX-M-group 1, CTX-M-group 2, and CTX-M-
group 9, and OXA-, SHV- and TEM-genes, by multiplex
PCRs using primers described by Dallenne et al. (2010). For
amplification, 3 ml of DNA extract (the same as was used
for phylogenetic typing) was mixed with 10 pmol of each
primer and 12.5 ml Qiagen Multiplex PCR mix (Qiagen
Benelux BV, Venlo, the Netherlands) in a final volume of
25 ml. Amplification conditions were as described by
Dallenne et al. (2010). PCR-products were analyzed on
agarose gel. To identify individual alleles, PCR-products of
the expected size (TEM: 800 bp, SHV: 713, CTX-M group
1:688 bp, CTX-M group 2:404 bp, CTX-M-group 9: 561 bp)
were treated with ExoSAP-IT (GE Healthcare, Hoevelaken,
the Netherlands) and sequenced using the same primers
used to generate the PCR-products, and BigDye Terminator
v3.1 Cycle Sequencing kit (Applied Biosystems, Bleiswijk,
the Netherlands). Thus obtained partial ESBL gene
sequences were compared with ESBL gene sequences in
the GenBank database and on the Lahey website (www.la-
hey.org/Studies). Partial sequence analysis allows identifi-
cation of particular clusters of homologous alleles, to
which, for sake of annotation, the lowest allele number
belonging to a cluster was assigned, e.g. blaCTX-M-1 includes
blaCTX-M-1 and blaCTX-M-61, see also Fig. 5).
2.4.1. Exclusion of within sample copy strains
Of all isolates for which ABR profiles, ESBL genotypes
and phylogenetic groups were determined, 10 were ‘twin’
isolates, i.e. isolates identical with respect to these

characteristics, originating from the same sample. Since it
was likely that these isolates were copy strains, they were
excluded from prevalence analyses, leaving 204 isolates
with assigned phylogenetic groups and 144 isolates with
determined ESBL-genotypes.
2.4.2. Multilocus sequence typing (MLST)
Among isolates for which ABR profiles, ESBL genotypes
and phylogenetic groups were determined, 37 were
identified that could be grouped into 15 sets of isolates
consisting of variants identical with respect to these
characteristics, obtained at different sites in a region on the
same sampling date. Of these 37 isolates, sequence types
were determined. For this purpose, seven house-keeping
genes, adk, fumC, gyrB, icd, mdh, purA and recA, were
amplified and sequenced as described by Wirth et al.
(2006). Primer sequences were obtained from the E. coli
MLST database website http://mlst.ucc.i.e./mlst/dbs/Ecoli.
For amplification, 2 ml of 10Â diluted DNA extract was
mixed with 200 pmol of each primer, 1Â PCR buffer
(Invitrogen, Bleiswijk, the Netherlands), 2.5 mM MgCl2
(Invitrogen), 200 mM dNTP mix (Invitrogen), and 1.25U
Taq polymerase (Invitrogen) in a final volume of 50 ml.
Amplification conditions were as follows: 5 min 95 8C,
followed by 35 cycles of 30 s 95 8C, 30 s 60 8C (adk, icd, mdh,
purA, recA) or 30 s 64 8C (fumC, gyrB), 45 s 72 8C, and a final
elongation step of 10 min 72 8C. PCR-products were
analyzed on agarose gel and PCR-products of the expected
size were treated with ExoSAP-IT (GE Healthcare, Hoeve-
laken, the Netherlands) followed by sequencing using the
same primers used to generate PCR-products, using BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems,
Bleiswijk, the Netherlands). Sequences were imported in
the E. coli MLST database website (http://mlst.ucc.i.e./mlst/
dbs/Ecoli) to determine MLST types.
3. Results
3.1. ESBL-producing E. coli in recreational waters and other
surface waters in the vicinity
ESBL-producing E. coli were detected in all four
recreational waters, in 44% (region B) to 100% (region D)
of samples (Table 1). Concentrations of ESBL-producing E.
coli ranged from 0.15 to 15 cfu/100 ml. For comparison,
total E. coli concentrations in the same samples ranged
from 23 to 5.2 Â 103
cfu/100 ml. ESBL-producing E. coli
represented 0.05–1% of the total E. coli population in
positive samples. In all regions, concentrations of ESBL-E.
coli at WWTP discharge points were comparable to that in
effluents, and on average 2- to 3-log 10 units higher than
that in the recreational waters (Fig. 2). In surface waters
upstream or not under the influence of the WWTPs
studied, average concentrations of ESBL-producing E. coli
were in the same range or slightly higher than those in the
recreational waters in the same area (Fig. 2).
3.2. Antibiotic resistance profiles of ESBL-producing E. coli
Next to cephalosporins and ampicillin (explained by
ESBL production), the majority of all ESBL-producing E. coli
from surface waters, including recreational waters, and
WWTP effluents were resistant to sulfoxisole (74%),
trimethoprim (67%), tetracycline (60%), and nalidixic acid
(60%). Resistance to ciprofloxacin (42%) and streptomycin
(49%) was also common, while resistance to gentamicin
(29%), chloramphenicol (19%), coamoxiclav (10%), and
cefoxitin (3%) was less frequently observed. None of the
isolates were resistant to the carbapenem antibiotics
imipenem and meropenem. Among ESBL-producing iso-
lates from recreational waters, resistance to (fluoro)qui-
nolones, aminoglycosides and tetracycline appeared lower
compared to isolates from wastewater and other surface
waters (Fig. 3a). Overall, 71% of isolates from WWTP
effluents, 63% of isolates from discharge points, 62% of
isolates from surface waters upstream and not under
influence of the investigated WWTPs, and 41% of isolates
from recreational waters were resistant to at least three
classes of antibiotics additional to beta-lactams, and
therewith defined as multidrug-resistant (MDR)
(Fig. 3b). In total, 14% of all ESBL-producing E. coli were
resistant to five (12%), six (1.8%) or even all seven (0.7%)
additional classes of antibiotics.
For each sampling region, the resistance phenotypes
of isolates resistant to one to four classes of antibiotics in
addition to beta-lactams were compared between
sampling sites (Fig. 4). Resistance phenotypes detected
in recreational waters were frequently, but not always,
detected in WWTP effluents and/or in surface waters
located upstream of recreational waters, including
surface water at WWTP discharge points and surface
water upstream of WWTPs. Isolates resistant to five or six
classes of antibiotics were not included in this analysis,
because there was hardly any variation in phenotypes
among such isolates (e.g. almost two-third of all isolates
resistant to five classes of antibiotics next to beta-
lactams were resistant to sulfanomides/trimethoprim,
(fluor)quinolones, tetracycline, aminoclycosides and
chloramphenicol).
Fig. 2. Concentrations ESBL-producing E. coli in surface water and
wastewater. Error bars represent the standard error of the means.
Numbers on top of the bars represent the number of samples on which
average concentrations were based. For region A, results from two sites
upstream of the WWTP have been pooled, for region C results from two
surface waters not influenced by studied WWTP have been pooled;
ns = not sampled; SW = surface water; n.u.i. = not under the influence of
studied WWTP.

3.3. ESBL-genotypes and phylogenetic groups
Almost two-third of 144 ESBL-producing E. coli
obtained from Dutch surface waters, including recreational
water, and WWTP effluents carried blaCTX-M-15 or blaCTX-M-1
(Fig. 5a). These two ESBL-genes were predominant in all
four regions, although blaCTX-M-1 was detected about two
times less frequently in region C compared to the other
three regions (Fig. 5b). For some ESBL-genotypes region-
specific patterns were observed, e.g. blaCTX-M-14 was more
prevalent in regions A and B, blaSHV-12 was more prevalent
in regions B and C, blaCTX-M-3 and blaCTX-M-9 were only
detected in region C, and blaCTX-M-32 and blaTEM-52 were
only detected in regions A and C. The least frequent
genotypes were blaCTX-M-2 and blaCTX-M-24 which were both
detected once (0.7% of all isolates). All ESBL-genotypes that
were detected in recreational waters were also detected in
WWTP effluents and/or surface waters located upstream of
the recreational waters (including discharge points and
waters upstream of WWTPs). This was also the case for
relatively infrequently observed and region-specific geno-
types, such as blaCTX-M-9, blaCTX-M-14, blaCTX-M-32, blaSHV-12,
and blaTEM-52 (Fig. 5b).
When ESBL-genotypes were compared within the
context of E. coli phylogenetic groups, it appeared that even
though the same ESBL-genes were detected at different sites
within a region, they were often present in diverse
phylogenetic backgrounds (Fig. 6). In a minority of cases
(n = 13) in regions A, C and D, E. coli of the same phylogenetic
group and carrying the same ESBL-genes were observed in
recreational waters as well as WWTP effluents or upstream
located surface waters. Of note, even though the majority of
ESBL-producing E. coli from surface water belonged to
phylogenetic groups generally associated with commensal
E. coli, also phylogenetic (sub)groups associated with
virulent strains, i.e. B2 and D2 (Anastasi et al., 2012; Duriez
Fig. 3. Resistance phenotypes of ESBL-producing E. coli from surface waters and wastewaters. Shown are the percentages of isolates resistant to each
antibiotic tested (A) and percentages of isolates resistant to different numbers of antibiotic classes besides beta-lactam antibiotics (B). N.U.I = not under the
influence of the studied WWTP (i.e. upstream of the WWTP and other connecting water bodies not under influence), D.P. = discharge point. The percentages
on top of the bars in (B) represent the percentages of multidrug resistant isolates.

et al., 2001; Jakobsen et al., 2010; Johnson et al., 2001; Picard
et al., 1999), were detected. In a randomly selected subset of
120 isolates with assigned phylogenetic groups, 32% of
wastewater isolates and 7.9% of isolates from surface water
including recreational water (and discharge points exclud-
ed) belonged to (sub)groups B2 or D2.
When including antibiotic resistance profiles, 15 sets of
isolates were identified that consisted of isolates identical
with respect to phylogenetic group, ESBL-genotype, the
presence of TEM- and/or OXA- non-ESBL-genes, and ABR
profile, obtained at different sites in a region on the same
sampling date (Table 2). In four cases (three for region A,
and one for region D), this match was observed for isolates
from recreational water and isolates from upstream
located surface waters and/or effluents.
3.4. Sequence types
The sequence types of 37 isolates making up 15 sets of
matching isolates were determined. In all cases, isolates
within sets were identical with respect to phylogenetic
group, ESBL-genotype (including TEM and/or OXA-non-
ESBL genes), ABR profile, and sequence type. Isolates from
four of 15 sets were identified as ST617 (Table 2). In one set,
ST617 isolates carried blaCTX-M-1, the ST617 isolates in the
other three sets carried blaCTX-M-15. ST617/CTX-M-15
isolates with identical ABR profiles were detected at two
different time-points in surface waters and WWTP effluents
in region A (Table 2). The remaining eleven sets contained
‘unique’ sequence types: ST10, ST58, ST69, ST86, ST90,
ST160, ST361, ST761, ST1722,ST2211 and one unknown. For
sixof thesesequence types, information on the phylogenetic
groups is available at the E. coli MLST website (http://
mlst.ucc.i.e./mlst/dbs/Ecoli) and/or has been described by
others (Alouache et al., 2014; Deng et al., 2011), in complete
agreement with our current findings: A/ST10, B1/ST58, D/
ST69, A/ST90, A/ST160, and A/ST617 (Table 2).
4. Discussion
During the bathing seasons of 2011 and 2012, ESBL-
producing E. coli were detected in four recreational waters
Quin-Amgl
Quin-BLca
Quin-Tet
Sulfa-Amgl
Sulfa-Ceph
Sulfa-Chl
Sulfa-Quin
Sulfa-Tet
Tet-BLca
Amgl
Chl
Quin
Sulfa
Tet
Quin-Amgl-Chl
Quin-Tet-Amgl
Quin-Tet-Chl
Sulfa-Amgl-BLca
Sulfa-Amgl-Chl
Sulfa-Quin-Amgl
Sulfa-Quin-Ceph
Sulfa-Quin-Tet
Sulfa-Tet-Amgl
Sulfa-Amgl-Chl-Ceph
Sulfa-Quin-Amgl-BLca
Sulfa-Quin-Amgl-Chl
Sulfa-Quin-Tet-Amgl
Sulfa-Quin-Tet-BLca
Sulfa-Quin-Tet-Chl
Sulfa-Tet-Amgl-BLca
Sulfa-Tet-Amgl-Chl
Region A Region B Region C Region D
8
6
2
0
4
Numberofisolates
UP EF DP RW EF DP DN RW
10
8
4
0
6
2
UP EF DP NIRW
UP EF DP RW
UP EF DP RW
UP EF DP RW UP EF DP RW
EF DP DN RW
UP EF DP RW
UP EF DP RW
EF DP DN RW
EF DP DN RW
UP EF DP RW
UP EF DP NIRW
UP EF DP NIRW
UP EF DP NIRW
10
8
4
0
6
2
10
8
4
0
6
2
12
14
Fig. 4. Antibiotic resistance profiles. Shown are the numbers of ESBL-producing E. coli isolates with indicated resistance profiles for isolates resistant to 1, 2, 3
or 4 antibiotics (rows 1–4 respectively), for each sample type and each region. For region A, isolates from two upstream sites have been pooled, for region C,
isolates from two connecting water bodies not under influence of the studied WWTP have been pooled. UP = upstream of WWTP; EF = WWTP effluent;
DP = discharge point; RW = recreational water; DN = downstream of WWTP; NI = connected surface water bodies not under influence of studied WWTP.

situated nearby and under the influence of WWTPs. A
substantial proportion of these ESBL-producing E. coli was
multidrug-resistant. Detection of ESBL-producing E. coli
appeared to be associated with relatively high concentra-
tions of total E. coli. Nevertheless, the proportion of ESBL-
producing E. coli relative to total E. coli numbers varied
among sites, as well as between time-points at the same
site, ranging from 0.05% to 1%. Overall, concentrations of
ESBL-producing E. coli were similar in both bathing
seasons, although in region B the frequency of detection
appeared lower in 2012 as compared to 2011. At this site,
the concentration of total E. coli was also lower in 2012
than in the previous year. A possible explanation may be
that in 2012 a small part of the lake adjacent to the WWTP
was dammed to retain sewage overflows (but not
effluents) which were therewith no longer discharged
A.
B.
24,31%
42,36%
1,39%
3,47%
0,69%
2,78%
9,72%
2,08%
0,69%
8,33%
4,17%
CTX-M-1
CTX-M-15
CTX-M-3
CTX-M-32
CTX-M-2
CTX-M-9
CTX-M-14
CTX-M-27
CTX-M-24
SHV-12
TEM-52
0%
20%
40%
60%
80%
100%
Total (n=45) Upstream
WWTP (n=17)
WWTP
effluent
(n=11)
Discharge
point (n=6)
RecreaƟonal
water (n=11)
%Isolates
Region A
CTX-M-1 CTX-M-15 CTX-M-32 CTX-M-14 SHV-12 TEM-52
0%
20%
40%
60%
80%
100%
Total (n=15) WWTP
effluent (n=3)
Discharge
point (n=5)
Downstream
WWTP (n=5)
RecreaƟonal
water (n=2)
%Isolates
Region B
CTX-M-1 CTX-M-15 CTX-M-14 CTX-M-24 SHV-12
0%
20%
40%
60%
80%
100%
WWTP (n=4)
WWTP
effluent
(n=17)
Discharge
point (n=17)
RecreaƟonal
water (n=9)
Surface
water N.U.I
(n=13)
%Isolates
Region C
CTX-M-1 CTX-M-3 CTX-M-15 CTX-M-32 CTX-M-9
CTX-M-14 CTX-M-27 SHV-12 TEM-52 CTX-M-2
0%
20%
40%
60%
80%
100%
WWTP (n=7)
WWTP
effluent (n=3)
Discharge
point (n=4)
RecreaƟonal
water (n=10)
%Isolates
Region D
CTX-M-1 CTX-M-15 CTX-M-14 CTX-M-27 SHV-12
n=144
Fig. 5. ESBL-genes detected in ESBL-producing E. coli from Dutch wastewater and surface water. Shown are the overall distribution of ESBL-genotypes in 144
isolates from wastewaters and different surface waters (A) and the distribution of ESBL-genotypes per sampling region and sample type (B). N.U.I. = not
under influence of the WWTP studied. Each indicated genotype represents a cluster of closely related, on the basis of partial sequence indistinguishable,
variants: ‘CTX-M-1’ = CTX-M-1/61, ‘CTX-M-2’ = CTX-M-2/20/44/56/59/97, ‘CTX-M-3’ = CTX-M-3/22/66, ‘CTX-M-15’ = CTX-M-15/28, ‘CTX-M-32’ = CTX-M-
32, ‘CTX-M-9’ = CTX-M-9/51, ‘CTX-M-14’ = CTX-M-14/21/83/90/113, ‘CTX-M-27’ = CTX-M-27/98, ‘CTX-M-24’ = CTX-M-24/65, ‘SHV-12’ = SHV-12/129,
‘TEM-52’ = TEM-52/92.

Fig. 6. Phylogenetic groups and ESBL-genes of ESBL-producing E. coli. Shown are the numbers of isolates with indicated ESBL genotypes in phylogenetic
backgrounds for different samples and for each region. Isolates from wastewater and from surface water at the discharge points have been pooled. *For
region C, surface water upstream of recreational waters encompasses surface waters upstream of WWTP as well as surfacewaters not under inﬂuence of the
studied WWTP.

onto the lake (unless in situations of extreme rainfall
causing spill-over of the dam).
All WWTP effluents contained ESBL-producing E. coli, as
did receiving surface waters at the points of discharge. At
these sites, ESBL-producing E. coli isolates can be assumed
to directly reflect the bacterial population present in
discharged effluents at that moment of sampling, although
a small proportion of isolates may have been derived from
upstream locations. In line with this assumption, concen-
trations of ESBL-producing E. coli at discharge points were
similar to that in effluents, and on average 1- to 2-log 10
units higher than concentrations upstream of the WWTPs,
or in connecting water bodies not influenced by the
investigated WWTPs. Considering an average concentra-
tion of 2 Â 102
–5 Â 102
ESBL-producing E. coli in 100 ml of
effluent, and an average daily discharge of 10,000–
35,000 m3
of effluent by the investigated WWTPs, approx-
imately 1010
–1011
ESBL-producing E. coli are discharged
daily per WWTP. Even though these findings demonstrate
that WWTPs contribute to the presence of ESBL-producing
E. coli in surface water, ESBL-producing E. coli were also
frequently detected upstream of the WWTPs, and in
connecting water bodies not influenced by the studied
WWTPs, implicating the existence of additional sources of
ESBL-producing E. coli. Moreover, since ESBL-producing E.
coli were detected in multiple water bodies and in different
regions in the Netherlands, these data suggest that
contamination sources are abundant. However, in a
recreational water located within approximately 200 m
of, but not connected to, recreational water D, ESBL-
producing E. coli were not detected at three time-points
during bathing season 2012 (data not shown), demon-
strating that at least some surface waters are only
infrequently, if at all, exposed to ESBL-producing E. coli
– containing fecal contamination sources.
The current study focused on discharged effluents of
nearby located WWTPs as possible source of ESBL-
producing E. coli in recreational waters, but did not
investigate the contribution of sewage overflows or more
remote WWTPs. Since overflows contain untreated sew-
age, they may be an important source of ESBL-producing E.
coli in surface water during heavy rainfall. Locations of
overflow exhausts in the investigated regions were not
mapped, however, both overflows and more remote
WWTPs may have contributed to the fecal contamination
in the investigated surface waters, in recreational waters as
well as at the other sampling sites. Additionally, animal
manure and droppings from pasture animals may have
contributed, since ESBL-producing E. coli are abundant in
Dutch food animals, in particular in broilers, but also in
pigs and veal calves (Nethmap_MARAN, 2013). Finally, also
feces of wild animals such as birds, may contribute to
ESBL-producing E. coli in surface water (Guenther et al.,
2011). With the exception of region C, all investigated
regions are located in rural areas, making it probable that
the ESBL-producing E. coli in the investigated surface
waters are of mixed human and animal origin.
ESBL-producing E. coli recovered from recreational
waters carried similar ESBL-genes, partially in the same
phylogenetic background, as ESBL-producing E. coli in
effluents and/or upstream located surface waters. Most
Table2
Characteristicsofsetsofidenticalisolatesobtainedfromdifferentsamplesinthesameregion.
RegionTime-pointPhylogenetic
(sub)group
ESBL
gene
Non-ESBLTEM/OXA
genea
MLSTABRProfileUpstream1b
Upstream2b
WWTPDischarge
point
Recreation
water
SWn.u.i
A1A1CTX-M-1TEMST617Am,Cx,Ge,St,Te,Ci,Na,Su,Tr++++Àn.s.
A1A1CTX-M-15OXAST617Am,Cx,Cz,Ge,Te,Ci,Na,Su,TrÀ++++n.s.
A1B1CTX-M-14ÀST86Am,Cx,St,Te,Ci,Na,Su,Tr,Ch+À+ÀÀn.s.
A1D1CTX-M-15TEM,OXAST69Am,Cx,Cz,St,Te,Su,Tr++ÀÀÀn.s.
A1B1CTX-M-1TEMST58Am,Cx,St,Te,Su,Tr+À+ÀÀn.s.
A2A1CTX-M-15ÀST90Am,Cx,Cz,Ci,NaÀ+À+Àn.s.
A2D1CTX-M-15TEMST1722Am,Cx,Cz,Ge+À+À+n.s.
A2A1CTX-M-15OXAST617Am,Cx,Cz,Ge,Te,Ci,Na,Su,Tr++++Àn.s.
A2A0CTX-M-1ÀST160Am,Cx,Su,TrÀ+ÀÀ+n.s.
C1A1CTX-M-15OXAST361Am,Cx,Cz,Ax,Ge,Ci,Na,Su,TrÀn.s.++ÀÀ
C1A1CTX-M-15ÀST10Am,Cx,NaÀn.s.++ÀÀ
C3A1CTX-M-15TEMST2211Am,Cx,Cz,St,Te,Su,TrÀn.s.++ÀÀ
C3B1CTX-M-15ÀUnknownc
Am,Cx,Cz,Te,Su,TrÀn.s.++ÀÀ
D2A1CTX-M-15OXAST617Am,Cx,Cz,Te,Ci,Na,Su,TrÀn.s.++Àn.s.
D2A1CTX-M-1ÀST761Am,Cx+n.s.ÀÀ+n.s.
a
IdentifiedusingPCR,typeswerenotdetermined.
b
ForregionAtwositesupstreamWWTPweresampled(seeFig.1).
c
Allelenumbersforhousekeepinggenesadk,fumC,gyrB,icd,mdh,purA,recAwere6,133,12,1,9,5,and7respectively.n.s.=notsampled;SWn.u.i=waternotundertheinfluenceofstudiedWWTP,
Am=ampicillin,Cx=cefotaxime,Cz=ceftazidime,Ge=gentamicin,St=streptomycin,Te=tetracycline,Ci=ciprofloxacin,Na=nalidixicacid,Su=sulfoxizole,Tr=trimethoprim.

notably, infrequently detected ESBL-genotypes – some-
times exclusively observed in certain regions – were also
detected in recreational waters, suggesting a relation
between variants in recreational water and variants
circulating in the region. In four cases, ESBL-producing E.
coli variants from recreational waters were identical to
those in wastewater and/or upstream located surface
water sites obtained on the same day, with respect to
phylogenetic group, ESBL-genotype ABR-profiles and
sequence types. In two of these four cases this concerned
variants from recreational water and wastewater. These
data may indicate that these four ESBL-producing E. coli
strains present in recreational waters originated from
WWTP effluents or from a source further upstream, and
migrated to the recreational waters with the current.
Alternatively, identical ESBL-producing variants present at
different sites may originate from different sources, which
could be the case when specific variants are widely
dispersed among humans and/or animals. Some of the
surface water and effluent E. coli sequence types, i.e. ST10,
ST58, ST69 and ST617, have previously been detected in
Dutch human and animal clinical isolates obtained, either
or not associated with the same ESBL-genes (Dierikx et al.,
2012; Leverstein-van Hall et al., 2011; Overdevest et al.,
2011; Reuland et al., 2013).
Even though the current study demonstrates a contri-
bution of WWTPs to the presence of ESBL-producing E. coli
in surface waters, their relative contribution to the
contamination of the recreational waters under study
remains undetermined. Only a small number of ESBL-
producing E. coli variants from recreational waters had
identical counterparts in effluents or at discharge points. In
most samples including recreational water samples,
however, a high diversity of ESBL-producing E. coli
phenotypes was observed. As a consequence, the odds of
detecting copy isolates from two different places in
connecting water bodies is small and dependent on the
number of isolates that are analyzed. In the current study
maximally 10 isolates were isolated and characterized per
sample, which considering the observed large variation in
phenotypes, may have resulted in an underestimation of
the level of accordance between isolates from wastewater
and recreational waters. Analysis of a larger number of
isolates per site, mapping and sampling of additional
contamination sources in the investigated area (i.e. over-
flows, other, more remote WWTPs, and animal manure),
and taking into account current velocities between
discharge points and recreational waters, are essential to
unequivocally determine the relative contributions of
WWTPs and other contamination sources to the presence
of ESBL-producing E. coli in recreational waters. Moreover,
for the purpose of tracing variants to specific sources, a
more thorough characterization of isolates would need to
be considered, including an additional method for typing of
the E. coli strains, such as pulsed-field gel electrophoresis
(PFGE), and analysis of the ESBL-carrying plasmids using
PCR based replicon typing (PBRT) (Carattoli et al., 2005;
Nemoy et al., 2005).
Based on obligatory measurements of fecal indicators E.
coli and intestinal enterococci of 2009–2011 in official
recreational waters performed by water boards, and
according to guidelines described in the European Bathing
Water Directive (Anonymous, 2006), the water quality of
the three official recreational waters was classified as
‘poor’ (region B), ‘acceptable’ (region A), or ‘good’ (region
C). As of 2013, ‘signal values’ are used to assess individual
samples for compliance; the signal value for E. coli is set at
1800 cfu/100 ml. Based on this value and our 2011 and
2012 measurements, non-compliance was observed only
at one time-point in recreational waters A and B (both in
2011). So, multidrug resistant ESBL-producing E. coli were
detected in recreational waters classified from ‘poor’ to
‘good’, at time-points when recreational waters complied
with bathing water legislation.
The rationale for investigation of ESBL-producing E. coli
in surface and recreational waters is twofold. Besides
determining the relative contribution of different contam-
ination sources for the purpose of intervention strategies, it
is necessary to determine the contribution of recreational
water (or other environment-related exposure) to the
overall community-associated exposure to ESBL-produc-
ing bacteria, relative to that of consumption of contami-
nated food or direct contact with animal or human carriers.
Considering that with the exception of people working at
farms, people may have a better opportunity to come into
contact with contaminated recreational water than with
livestock, the detection of ESBL-producing E. coli in
approved recreational waters suggests that recreational
waters should be considered as a potential exposure route
of these bacteria.
Conflict of interest
None to declare.
Role of the funding source
The funding source has had no involvement in study
design, the collection, analysis and interpretation of data,
the writing of the manuscript, or the decision to submit for
publication.
Acknowledgements
The authors are thankful to Arieke Docters van
Leeuwen, Gretta Lynch, and Christiaan Veenman for
excellent technical assistance, to the water boards
‘Wetterskip Fryslan’, ‘Hoogheemraadschap Rijnland’,
‘Waterschap Rijn en IJssel’, and ‘Rijkswaterstaat IJssel-
meergebied’, for providing essential information regarding
WWTPs and recreational waters in the respective areas,
and to the people working at the WWTPs for providing
effluent samples. This study was financed by the Dutch
Ministery of Infrastructure and the Environment.
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