Science of the Total Environment 650 (2019) 2141–2149

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Science of the Total Environment

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

Assessing microbial contamination and antibiotic resistant bacteria using

zebra mussels (Dreissena polymorpha)
Maria Alexandra Bighiu a,⁎, Anna Norman Haldén b, Willem Goedkoop a, Jakob Ottoson c
a
Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences (SLU), Sweden
b
Department of Biomedical Sciences and Veterinary Public Health, SLU, Sweden
c
Department of Risk Benefit Assessment, National Food Agency, Sweden

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

• Faecal contamination of water causes

severe health effects worldwide.
• We studied the uptake of faecal
indicator bacteria by zebra mussels.
• The concentration of bacteria in mussels
was 132 times higher than in water.
• Antibiotic resistance was more
prevalent among bacteria from mussels,
than water.
• Zebra mussels are suitable for monitoring
pathogens and detecting antibiotic
resistance.

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

Article history: Aquatic pollution with faecal bacteria and subsequent consumption of contaminated water or food is a world-
Received 4 July 2018 wide issue that causes severe health effects (e.g. meningitis, salmonellosis, dysentery). In addition, the excessive
Received in revised form 20 September 2018 use of antibiotics in animal husbandry and human medicine has enhanced the selective pressure on pathogenic
Accepted 24 September 2018
bacteria, further increasing human health risks and detrimental effects on natural microbial communities. This
Available online 25 September 2018
urges the need to monitor faecal contamination using a time-integrated approach, as grab water samples can
Editor: Henner Hollert miss pathogen peaks. We tested the ability of zebra mussels (Dreissena polymorpha) to take up and depurate fae-
cal indicator bacteria such as Escherichia coli and intestinal enterococci. Furthermore, we quantified the frequency
Keywords: of antibiotic resistant bacteria in water and mussels both in controlled laboratory tests and under in situ condi-
Faecal tions downstream of a sewage treatment plant (STP). Laboratory results show that bacterial indicators in mussels
Pathogen were 132 times higher than their concentration in water, and that mussels retained bacteria up to 2 days after
In situ pulse exposure. Field results show decreasing bacterial concentrations in both water and mussels downstream
Escherichia coli the STP, with maximum E. coli concentrations ranging 173–9 cfu mL−1 in water and 2970–330 cfu g−1 in mussels.
Enterococcus
Similarly, enterococci ranged 59–4 cfu mL−1 and 1450–240 cfu g−1 in water and mussels, respectively. High pro-
Salmonella
portions of antibiotic resistant E. coli were found in mussels (72%) and water (65%), and slightly lower proportion
of resistant enterococci was found in mussels (47%) and in water (34%). Moreover, 33% of the bacteria isolated
from mussels were resistant to multiple antibiotics, which emphasizes that resistance is a common feature in sur-
face waters and highlights the need for safe water management. Our results show that zebra mussels provide an

⁎ Corresponding author at: Box 7050, 750 07 Uppsala, Sweden.

E-mail address: maria.bighiu@slu.se (M.A. Bighiu).

https://doi.org/10.1016/j.scitotenv.2018.09.314
0048-9697/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
2142 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149
efficient, time-integrating tool for quantifying faecal indicators, including resistant and multidrug resistant
bacteria.
© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
1. Introduction
Faecal contamination is a global problem, resulting in unsafe drink-
ing water (WHO, 2002) and water- and foodborne diseases such as
meningitis, gastroenteritis, salmonellosis, hepatitis and dysentery
(WHO, 2015a). For example, diarrheal diseases are estimated to account
for 1.4 million deaths each year worldwide, thus being one of the major
causes of human mortality and morbidity (GBD, 2017). More than 100
types of faecal pathogens have been identified, including bacteria, vi-
ruses and parasites (Ottoson et al., 2005) which are associated with
and dispersed by human and animal excrements. The abundance of fae-
cal pathogens in water can have large and rapid fluctuations and con-
tribute to a widespread human exposure to contaminated water.
Contaminated food may cause severe health effects, high economical
losses, and shortages of safe food supplies. Therefore, knowledge of
the source water quality, in particular the concentrations of reference
pathogens or indicators, is essential for designing water safety plans
and for reaching public health protection targets (WHO, 2005). As path-
ogens originating from different sources can be transmitted faecally-
orally from water (Pandey et al., 2014), faecal pollution is best moni-
tored by concentrations of indicator bacteria. E. coli is the most com-
monly used indicator, but as it is more sensitive to environmental
stress than several enteric viruses and protozoan cysts (Yates, 2007),
additional indicators such as intestinal enterococci are also commonly
used (e.g. European Union Bathing Water Directive, 2006/7/EC).
Also the presence of antibiotics in the environment, including source
waters and their potential risks to human and environmental health
(Costanzo et al., 2005), is a major concern for many governments,
drinking-water regulators, water suppliers, and the public. For example,
12.7 million kg of antibiotics were consumed in the EU in 2014, with
70% applied in animal husbandry (ECDC et al., 2017). Similarly,
17.2 million kg antibiotics were sold in the US in 2013, while the esti-
mated use in China in 2013 was almost tenfold higher (Wang et al.,
2015). Between 30% and 90% of used antibiotics are excreted unchanged
or as active metabolites into the environment via urine and faeces
(Jjemba, 2006; Lienert et al., 2007). Despite some reduction during
wastewater treatment, antibiotics are still entering surface waters (Xu
et al., 2007), and can create a selective pressure for resistance among
pathogens (Gullberg et al., 2011; Levy and Marshall, 2004). Subse-
quently, this can have detrimental effects on human health as it ham-
pers the efficient treatment of many infections (Levy and Marshall,
2004; White et al., 2002). WHO's Global action plan on antimicrobial re-
sistance underlined the need for an effective “one health” approach in-
volving coordination among numerous international sectors and actors
(WHO, 2015b). Besides the human health risk, antibiotics affect micro-
bial biodiversity by selecting the resistant species and consequently
contaminating the environment by dissemination of antibiotic resistant
genes (Martinez, 2009).
In order to improve the detection of pathogenic bacteria and antibi-
otic resistance, time-integrated methods are needed, as water grab sam-
ples provide a poor chance of detection (Moles and Hale, 2003). Here
we evaluated the suitability of zebra mussels (Dreissena polymorpha)
as bioindicators of pathogenic and antibiotic-resistant bacteria in fresh-
water recipients. We did so by quantifying the uptake and retention of
faecal indicator bacteria in mussels under laboratory and field condi-
tions. We also assessed antibiotic resistance among bacteria isolated
from sewage effluent water and mussels. We hypothesized (1) that
both the concentration of bacterial indicators and the frequency of anti-
biotic resistant bacteria would be higher in mussels than in the water
and (2) that the mussels would retain the bacteria a few days after a
(http://creativecommons.org/licenses/by/4.0/).
pulse exposure. Moreover, (3) we expected a decline in bacterial con-
centration and frequency of antibiotic resistance downstream from
the STP.
2. Materials and methods
2.1. Test organisms
Zebra mussels are freshwater bivalves native to the Black and
Caspian Seas (Ludyanskiy et al., 1993), and are widely distributed across
Europe and North America (Strayer, 1991). They are epifaunal and have
a life span of about 3–5 years (Strayer and Malcom, 2006). The females
are highly fecund, and release up to 90,000 eggs ind−1 y−1 (Schneider,
1992). Zebra mussels feed on suspended particles in the range of
0.7–200 μm (Sprung and Rose, 1988; Winkel and Davids, 1982) at
high filtration rates, 46 mL mg−1 h−1 (Fanslow et al., 1995). Hence,
zebra mussels take up large amounts of suspended particles and deposit
faeces and/or pseudofaeces on the bottom (Mackie, 1991). Hence,
through their filter-feeding behaviour, zebra mussels are readily ex-
posed to both particle-associated and dissolved contaminants. They
are good for biomonitoring contaminants (Cope et al., 1999), as they
are sessile, highly abundant, widely spread, easy to collect and have a
low susceptibility to serious diseases (Karatayev et al., 2002).
We used two bacterial indicators, E. coli and Enterococcus spp. that
both live in the intestinal tract of warm-blooded animals and therefore,
their presence in water bodies is usually associated with faecal contam-
ination. E. coli are aerobic Gram-negative, rod-shaped bacteria (Ø 0.5
μm) (Britannica). Enterococci are facultative-anaerobic, Gram-positive
bacteria with ovoid shape (Ø 0.5–1 μm) (Zhou and Li, 2015).
2.2. Uptake and elimination of bacteria by mussels in laboratory
experiments
Zebra mussels (shell length 15.8 ± 4.2 mm) were collected in winter
from Lake Erken, Sweden (59°50′15.6″N 18°38′06.1″E) and transported
back to the laboratory in cold lake water (approximately 5 °C). The mus-
sels were acclimatized to experimental temperatures for 21 days, dur-
ing which the temperature was increased successively from 5 (in situ)
to 18 °C, and mussels were fed every 4th day with ground Tetraphyll®.
During the acclimation period lake water was aerated and gradually re-
placed by artificial lake water, i.e. M4-medium prepared by adding nu-
trients to Milli-Q water (OECD Guideline 202), the medium used in
laboratory experiments.
All aquaria, beakers, nets, and aeration hoses were autoclaved prior
to each experiment. Laboratory experiments were done in triplicates
at 17.5 ± 0.5 °C (mean ± standard deviation) with a photoperiod of
16:8 h and constant aeration throughout the experiments. Mussels
were incubated in 25-L glass aquaria, placed on stainless-steel nets
that separated them from accumulating (pseudo)faeces underneath
the net, thus preventing re-ingestion of faecal material. Only mussels
that showed an active filtering behaviour were sampled at each time
point.
We studied the mussels' uptake and elimination of faecal indicator
bacteria using pure cultures, as well as a cocktail of bacteria (sewage ef-
fluent) in order to better understand the filtering behaviour of the zebra
mussels, and thus assess their suitability as biomonitoring organisms.
Hence, we gradually increased the complexity and relevance of the ex-
posure scenarios, from laboratory experiments, to the field experiment.
The overview of the 4 laboratory experiments is given in Table 1.
 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149
Table 1
Overview of the laboratory experiments.
Experiment Objective Source of bacteria
1 Uptake Laboratory culturea
2 Depuration Laboratory culturea
3 Uptake Sewage effluentb
4 Depuration Sewage effluentb
a
104 cfu mL−1 E. coli and E. faecalis.
b
10-Fold dilution of sewage effluent.
In the first experiment we quantified the uptake of bacteria in mus-
sels using pure cultures. Approximately 32 g mussels (or 32–39 individ-
uals) were placed in each 25 L aquarium containing 10 L of aerated
artificial lake water (M4). Escherichia coli (SLV082) and Enterococcus
faecalis (SLV051), cultured in BHI broth (Brain Heart Infusion, CM1135
Oxoid, UK) at 37 °C overnight, were added to experimental units to a
final density of 104 CFU (colony forming units) mL−1, which corre-
sponds to the concentrations measured in sewage effluent (Kay et al.,
2008). The mussels were then allowed to feed for 60 h on this bacterial
suspension and samples of water (200 mL each) and mussels (~3 g,
i.e., 2–6 individuals) were taken after 0, 2, 4, 6, 8, 12, 24, 48 and 60 h
for analysis of bacteria concentrations.
In the second experiment we quantified the elimination rate of cul-
tured bacteria by mussels. Using the same set-up as described above for
the first experiment, mussels were allowed to feed on bacteria for 8 h,
then rinsed with deionized water, and transferred into 1 L beakers con-
taining clean M4-medium for 60-h of depuration. Each beaker
contained a nylon net to keep the mussels separated from their depos-
ited faeces. During depuration, samples of mussels and water were
taken every 12 h for analysis of bacterial concentrations.
In the third experiment, following the set-up described above, we
quantified the mussels' uptake of bacteria from sewage effluent water
from the Uppsala Sewage Treatment Plant (STP), Kungsängsverket.
Mussels were exposed to a 10-fold dilution of the effluent (as previously
shown to be a suitable dilution by Selegean et al., 2001) in oxygenated
M4 media for 48 h. After 0, 2, 4, 6, 8, 12, 24, 36 and 48 h samples of
water and mussels were collected for determination of bacterial
concentrations.
In the fourth experiment, following the set-up described above, we
exposed the mussels to the sewage effluent dilution for 2 h. After this
uptake phase, the mussels were rinsed with deionized water and placed
in the depuration beakers. Water and mussel samples for bacterial enu-
meration were taken after 2, 4, 6, 12, 24, 36 and 48 h to quantify their
elimination rate of faecal indicator bacteria.
2.3. Uptake during in situ exposure
Adult zebra mussels (average shell length 20.2 ± 3.6 mm) were col-
lected from Lake Ekoln, Sweden in April 2012 (59°46′28.4″N 17°38′
39.0″E) and depurated for 9 days in sterile lake water, until no faecal in-
dicators were found in a tissue sample (i.e., 7.6 g ww). Approximately
100 mussels (90 g) were placed in duplicate metal cages (10 × 10
× 10 cm) at 0.5 m depth in the river Fyrisån at the outlet of the STP, at
an upstream site (120 m), and 4 sites downstream the STP (at 200,
500, 1000 and 1500 m) (Fig. 1). The Uppsala STP (www.
uppsalavatten.se, 59°50′39.1″N 17°39′35.6″E) processes annually
some 20 million m3 of sewage from the town of Uppsala (~120,000 in-
habitants and including a major regional hospital). The river Fyrisån is
a well-buffered (annual mean alkalinity 2.95 meq L−1), nutrient-rich
(annual mean total-P = 57 μg L−1) river that drains a 2006 km2 large
catchment predominated by forest (65%) and agricultural land use
(25%).
From each site, samples of water (200 mL) and mussels (6.74 ±
1.18 g, i.e., 4–9 individuals) were collected after 1, 2, 5, 7, 12 and 19 d
for the quantification of faecal indicators. Additional samples of water
(300 mL) and mussels (~25 g) for the isolation of Salmonella spp. were
2143
taken after 5, 12 and 19 d. On each sampling occasion, water tempera-
ture and pH were recorded at each site. Rainfall and discharge data
Duration (h) was available from the Swedish Meteorological and Hydrological Insti-
60
tute (SMHI).
60
48 2.4. Bacterial enumeration
48
Sampled, living zebra mussels were thoroughly rinsed with deion-
ized water and homogenized (i.e., including shells) using a blender.
The homogenates were diluted 10:1 with neutralized bacteriological
peptone water (SPW, LP0034 Oxoid, UK), spread on selective media,
and incubated 24 h on MacConkey agar (CM0115 Oxoid, UK) at 44 °C
for E. coli and 48 h on SlaBa agar (CM 0377 Oxoid, UK) at 44 °C, accord-
ing to the standard by the Nordic Committee on Food Analysis No. 68,
5th ed. (2011) for Enterococcus spp. The limit of detection for this anal-
ysis is 10 cfu g−1.
Water samples were diluted serially with SPW and a volume of
100 mL was filtered using a MicroFunnel™ 0.45 μm filter funnels
(PALL). The filter membranes were placed on selective agar and incu-
bated for 24 h on Lactose TTC with tergitol-7, (54232 Fluka analytical)
at 44 °C, according to ISO 9308-1 for E. coli and for 48 h on SlaBa at 44
°C, according to ISO 7899-2 for Enterococcus spp. At the end of the incu-
bation period, colonies formed by each faecal indicator were enumer-
ated and expressed as cfu g−1 (isolates from mussels) and cfu mL−1
(isolates from water). The limit of detection for this method is
0.01 cfu mL−1.
In the experiments with sewage effluent, it was necessary to further
confirm the bacterial colonies that differed in shape, colour or texture
from the typical ones. This was done by culturing on blood agar (SVA -
National Veterinary Institute, Sweden) at 37 °C for 24 h and by
performing biochemical tests – API 20E (Biomérieux) for E. coli and
sorbose tests for Enterococcus spp. We refer to enterococci as
‘E. faecalis’ for isolates from experiments with pure cultures and to ‘En-
terococcus spp.’ for isolates from experiments with sewage effluent or
the river water. One colony from each water or mussel sample was dis-
persed in 1.1 mL BHI with 17% glycerol and stored at −70 °C for antibi-
otic resistance testing.
In addition to E. coli and Enterococcus spp., water and mussel samples
from the field experiment were also analysed for Salmonella spp. For
this, 300 mL water samples were filtered (MicroFunnel™ 0.45 μm,
PALL) and the filters were incubated in 50 mL of buffered peptone
water (BPW, CM509 Oxoid, UK) at 37 °C overnight, according to ISO
19250. Salmonella spp. in mussels was analysed according to the Nordic
Committee of Food Analysis No. 187 (2007). Briefly, 25 g of mussels
were homogenized, diluted tenfold with BPW, and incubated at 37 °C
overnight. On the next day, 100 μL of each homogenate was transferred
to MSRV plates (Modified Semi-solid Rappaport Vassiliadis medium,
CM1112 Oxoid, UK) and incubated for 24 h at 42 °C. The plates showing
visible growth needed further confirmation by culturing on brilliant
green – BG (CM0329 Oxoid, UK) and xylose lysine desoxycholate -
XLD agars (CM0469 Oxoid, UK) for 24 h at 37 °C. These test are qualita-
tive, i.e. just score the presence or absence of Salmonella spp.
2.5. Assessment of antibiotic resistance
The resistance of faecal indicator bacteria to antibiotics was tested
for isolates both from the laboratory experiment with sewage effluent
(81 samples) and from the field experiment (71 samples), as well as
for all Salmonella spp. isolates (9 samples). These tests followed the
standards for microdilution of Cockerill and Clinical and Laboratory
Standards Institute (2015). VetMic™ GN-mo, VetMic™ E-cocci and
VetMic™ Salmonella microdilution panels (SVA, Sweden) were used ac-
cording to the instructions given by the manufacturer. Bacteria were
cultured on blood agar (SVA, Sweden) at 37 °C overnight. On the next
day, 3–5 colonies were suspended in 5 mL Mueller Hinton broth
(CM0405 Oxoid, UK) and incubated for 3 h at 37 °C, and diluted 1000
2144 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149
Fig. 1. Map showing the location of the experimental sites along the river Fyrisån at 120 m upstream (Site 1), at the outlet (Site 2), and 200, 500, 1000 and 1500 m (Sites 3–6) downstream
of the sewage treatment plant.
times. Then, 50 μL of this suspension was placed in each of the 96 wells
of the VetMic™ plates, covered with tape and incubated at 37 °C for
16–18 h. The antibiotics contained in the VetMic™ plates are listed in
Table 2. After the incubation, antibiotic-resistant bacteria were detected
in wells displaying visible growth above a specific level (i.e., MIC, mini-
mum inhibitory concentration). Information on MICs was obtained
from the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) (www.escmid.org). Isolates resistant to one or more antibi-
otics were classified as resistant whereas those resistant to ≥3 antibiotic
classes were judged multi-drug resistant (MDR), as suggested by
Schwarz et al. (2010).
2.6. Data analysis
JMP PRO v. 13 (SAS Institute Inc.) was used for statistical analyses.
One-tailed paired t-tests (α = 0.025) were applied to analyse whether
bacterial concentrations in mussels were higher than those in water. For
field experiment data, repeated measures ANCOVAs were used to test
for the effect of sites, exposure time, as well as their interaction, on bac-
terial concentrations in mussels and in water, respectively. Normality
and homoscedasticity of residuals were evaluated using normal QQ
plots and residual plots. Post-hoc pairwise, multiple comparisons were
done with Tukey HSD, while Pearson's correlations were run to evaluate
relationships between the two bacterial indicators at each site, as well
as between bacterial concentrations and environmental parameters
(i.e. as rainfall, river discharge, water temperature, pH). All results are
given as means ± standard deviations.
3. Results
3.1. Uptake and depuration
Zebra mussels showed a high capacity to take up bacteria in all lab-
oratory experiments. In uptake tests with pure cultures, the
 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149 2145

Table 2 mussels and water were consistently higher at the STP outlet than at
Cut-off values (in mg L−1, SVARM, 2016) of antibiotics contained in the VetMic™ plates for the upstream site, i.e., 23- and 57-fold higher E. coli concentrations, re-
each of the two bacterial indicators, E.coli and Enterococcus spp., as well as the pathogen
Salmonella enterica. N/A is not available because the antibiotic was not included in the
spectively. Similarly, faecal indicator concentrations in mussels and
plates. water at the STP-outlet were between 9- and 15-fold higher than
those at the downstream sites (Fig. 4, Table 3). On average, zebra mus-
Antibiotic tested E. coli E. faeciuma S. enterica
sels were 16-fold higher in E. coli and 39-fold higher in enterococci than
Ampicillin (Am) 8 4 8 ambient water. Concentrations of these two indicators found in mussels
Bacitracin (Ba) N/A 32 N/A
were similar, though they were only significantly correlated to each
Cefotaxime (Ctx) 0.25 N/A 0.5
Ceftazidime (Caz) 0.25 N/A N/A other at one site, namely the STP outlet (r = 0.83, p = 0.0008). Overall,
Chloramphenicol (Cm) 16 32 16 the concentrations of indicator bacteria in the water were significantly
Ciprofloxacin (Ci) 0.06 N/A 0.06 correlated to those in the mussels (r = 0.64 and 0.63, p b 0.0001 for
Colistin (Cs) 2 N/A N/A E. coli and Enterococcus spp., respectively).
Erythromycin (Em) N/A 4 N/A
Florfenicol (Ff) 16 N/A 16
Average water temperature during in situ exposure was 11.6 ± 1.4
Gentamicin (Gm) 2 32 2 °C and increased by 2° by the end of the 3 weeks of the study. E. coli con-
Kanamycin (Km) 8 1024 16 centrations in the water and in mussels (r = 0.6 and 0.42, p ≤ 0.0002 for
Linezolid (Lz) N/A 4 N/A both) correlated positively with water temperature, as did water con-
Nalidixic acid (Nal) 16 N/A 16
centrations of Enterococcus spp. (r = 0.55, p b 0.0001). Faecal indicator
Narasin (Na) N/A 2 N/A
Streptomycin (Sm) 16 128 16 bacteria in mussels or water did neither correlate with rainfall nor with
Tetracycline (Tc) 8 4 8 discharge. Water pH was significantly lower at the STP outlet (site
Trimethoprim (Tm) 2 N/A 2 2) than at all other sites (p b 0.0001 for all comparisons) and negatively
Vancomycin (Va) N/A 4 N/A correlated with the bacterial indicator concentrations in water and
Virginiamycin (Vi) N/A 4 N/A
mussels (r = −0.56 and −0.80, for E. coli in mussels and water, respec-
a
Note that the species of enterococci was not determined in this study, so we used the tively, p b 0.0001 for both and r = −0.41 and −0.74, p = 0.0003 and p b
cut-off values for E. faecium, as this is the most common in the studied area.
0.0001 for Enterococcus spp. in mussels and water, respectively).
Screening for Salmonella spp. showed positive results for 17% and
concentration of E. faecalis was almost 100-times higher in mussels 28% of the mussel and water samples, respectively (total 42 samples
(cfu g−1) than in water (cfu mL−1) (Fig. S1), while the equivalent for analysed). Mussels and water collected on all three occasions at the
E. coli was approximately 5 times (p b 0.0001 for both indicators). Inter- STP outlet were positive for Salmonella. Moreover, this pathogen was
estingly, the mussels showed a 20-fold higher assimilation of E. faecalis also detected in water samples from sites 4 and 3 (Fig. 1) on days 12
than of E. coli, despite similar average concentrations of E. coli and and 19, respectively.
E. faecalis in the water (i.e., 1476 and 1404 cfu mL−1, respectively). In
the depuration experiment, the highest elimination of bacteria from 3.2. Antibiotic resistance
the mussels occurred within the first 12 h of depuration, when concen-
trations decreased 42- and 62-fold for E. coli and E. faecalis, respectively. About half of all the faecal indicator bacteria isolated during the lab
After 60 h of depuration, 2.28 and 2.94 log reduction occurred in the experiment with sewage effluent showed antibiotic resistance. As
concentration of E. coli and E. faecalis, respectively, and implying an al- much as 73% of the E. coli isolates from mussels and 63% of those from
most 100% depuration for both (Fig. S2). In sewage effluent water the water were resistant against at least one antibiotic (Fig. 5). E. coli iso-
mussels showed a maximal uptake of both faecal indicators during the lated from mussels were most frequently resistant against tetracycline
first 2 h, i.e. 38–51% of the initial concentration was taken up. This re- and trimethoprim, while those isolated from water were resistant
sulted in concentration of E. coli and Enterococcus spp. in mussels that mainly against ampicillin and streptomycin. MDR colonies of E. coli
were 8- and 37-fold higher, respectively, than their water concentra- were found both in mussels (47%) and water (15%). Enterococcus spp.
tions (Fig. 2). The last depuration experiment showed a 0.94 and 1.62 showed a high susceptibility to antibiotics, namely 56% of those isolated
log reduction in the mussel concentrations of E. coli and enterococci, re- from mussels and 57% of the isolates from water. Additionally, MDRs
spectively (i.e., 83 and 95% depuration) (Fig. 3). were found in 19% and 4% of mussel and water samples, respectively.
In the field experiment, mussels downstream of the STP reached Most of the Enterococcus spp. colonies were resistant against tetracy-
maximum E. coli concentrations (i.e., 1400 cfu g−1) during the first cline and erythromycin.
24 h and maximum of enterococci (i.e., 360 cfu g−1) after 5 days. Bacte- In our field experiment, 68% of the E. coli colonies tested were resis-
rial concentrations in mussels and water then gradually decreased over tant against at least one antibiotic, while all E. coli colonies were suscep-
time at all sites, except for site 2 where an increase for both indicators in tible to chloramphenicol. All E. coli isolated from water were susceptible
mussels was noticed over time. In addition, enterococci in water col- to florfenicol and all those from mussels were susceptible to gentamicin.
lected from sites 3–6, increased slightly towards the end of the study Half of the enterococci isolated from mussels were resistant against at
(Fig. S3). As expected, the faecal indicator concentrations in both least one antibiotic, while those from water were resistant only against

Fig. 2. Uptake of E. coli and Enterococcus spp. from sewage effluent dilution by zebra mussels. Mean concentrations (±SD); Cfu = colony forming unit. N = 3 per time point and sample
type.
2146 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149

Fig. 3. Depuration of zebra mussels in still water (M4) after 2 h exposure to sewage effluent dilution. Mean concentrations (±SD); Cfu = colony forming unit. N = 3 per time point and
sample type.

tetracycline (25%). The highest frequency of resistant Enterococcus spp.
colonies in mussel samples, i.e. 33%, was found for tetracycline. As in the
lab experiment, MDRs prevailed in the mussel samples more than in
water samples, namely 35% (mussels) and 17% (water) for E. coli and
33% for enterococci isolated from mussels; no MDR enterococci were
found in water. Site 2 (the STP outlet) contained the highest frequency
of resistant E. coli and Enterococcus spp. for both water and mussel sam-
ples, followed by sites 3 and 6. The proportions of resistant isolates were
similar between the field experiment and the laboratory experiment
with sewage effluent, with the exception of enterococci isolated from
water. In this case, the isolates from the field experiment were only re-
sistant against tetracycline, whereas the isolates from the laboratory ex-
periment were resistant to 7 additional antibiotics as well (Fig. 5).
Salmonella spp. isolates were generally susceptible to most antibi-
otics tested (10 out of 12). Only two isolates from mussels and one
from water were resistant against streptomycin, and one isolate from
mussels and one from water were resistant against gentamycin (data
not shown). No MDR Salmonella spp. were detected.
4. Discussion
This study shows the rapid uptake of both E. coli and Enterococcus
spp. by zebra mussels from pure cultures and sewage effluent. Our find-
ings further show that, even after a short pulse exposure, bacteria can
persist in mussels for up to 48 h. This implies that mussels provide a
time-integrated measure of the water concentrations of bacterial indi-
cators, even several days after peak exposures. In addition, the observed
higher frequency of antibiotic resistant (including MDR) bacteria in the
mussels than in the water highlights the advantages of using zebra mus-
sels for monitoring water quality.
4.1. Uptake and depuration
Zebra mussels showed a high capacity to filter and retain bacteria
from both pure cultures and sewage effluents, with maximum levels
reached already after 2 h exposure in the laboratory. Similar rapid up-
take capacities in mussels have been reported for E. coli in previous
studies with zebra mussels (Selegean et al., 2001) and for enterococci
with blue mussels (Roslev et al., 2009). Moreover, uptake of E. coli by
mussels in our sewage effluent study is well in alignment with the re-
sults of Mezzanotte et al. (2016), where a 1.5 log uptake by zebra mus-
sels was reached after 4 h. In our first experiment with pure cultures
filtration rates decreased after 24 h, while our third experiment with
sewage effluent showed a slight increase in the filtration of E. coli after
48 h. This lower filtration rate in our first experiment is likely due to
the higher bacterial concentrations, which potentially have irritated
the mussels' tactile receptors and negatively affected filtration
(Morton, 1971).
In laboratory experiments, mussel uptake of enterococci was
1.2–34-fold higher than that of E. coli, thus corroborating results for

Fig. 4. Mean concentrations (±SD) of indicator bacteria – E. coli (upper panel) and Enterococcus spp. (lower panel) isolated from mussels (full bullets) and water (empty bullets) from the
river Fyrisån. Error bars show standard deviation obtained from duplicate cages at different sampling occasions (N = 12 per site for mussels and 6 for water); cfu = colony forming unit.
 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149 2147

Table 3
A) Output of the ANOVAs for testing the effects of site and exposure time on the concentration of bacterial indicators in mussels (repeated measures) and water. B) Output of the post hoc
Tukey HSD tests showing differences in bacteria concentrations between sites. Statistical significance is indicated as follows: **** for p ≤ 0.0001, *** for p ≤ 0.001, ** for p ≤ 0.01, and * for p ≤
0.05. The interaction between Site and Time for water samples did not improve the models and was not significant for any of the indicators, and hence it was removed.

A)

E. coli Enterococci

Effect p value F ratio df p value F ratio df

Mussels
Site 0.0016 17.58 5 0.0032 13.59 5
Time 0.2814 1.18 1 0.0464 4.16 1
Site ∗ time 0.8436 0.40 5 0.0022 4.32 5
Water
Site b0.0001 63.66 5 b0.0001 30.59 5
Time 0.5062 0.45 1 0.6195 0.25 1

B)

E. coli mussels E. coli water Enterococci mussels Enterococci water

Site −Site Difference Site −Site Difference Site −Site Difference Site −Site Difference

2 1 2*** 2 1 1.82**** 2 1 0.97** 2 1 1.8****

2 6 1.28** 2 4 1.24**** 2 6 0.63* 2 6 1.26****
2 4 1.24** 2 6 1.21**** 2 5 0.58* 2 5 1.21****
2 5 1.22* 2 5 1.16**** 4 1 0.57* 2 4 1.17****
3 1 1.06* 2 3 1.13**** 2 3 0.56* 2 3 1.05****
2 3 0.94* 3 1 0.69**** 3 1 n.s. 3 1 0.76***
5 1 n.s. 5 1 0.66**** 2 4 n.s. 4 1 0.64**
4 1 n.s. 6 1 0.61**** 5 1 n.s. 5 1 0.59**
6 1 n.s. 4 1 0.58**** 6 1 n.s. 6 1 0.54*
3 6 n.s. 3 4 n.s. 4 6 n.s. 3 6 n.s.
3 4 n.s. 5 4 n.s. 4 5 n.s. 3 5 n.s.
3 5 n.s. 3 6 n.s. 4 3 n.s. 3 4 n.s.
5 6 n.s. 5 6 n.s. 3 6 n.s. 4 6 n.s.
4 6 n.s. 3 5 n.s. 5 6 n.s. 5 6 n.s.
5 4 n.s. 6 4 n.s. 3 5 n.s. 4 5 n.s.

other mussels (Li et al., 2010; Marino et al., 2005). Olalemi et al. (2016),
however, reported higher uptake of E. coli than enterococci in Mytilus
edulis, illustrating that uptake rates of bacterial types differ among bi-
valve species, and emphasizing the need to use more than a single bac-
terial indicator.
The need for multiple faecal indicators is further highlighted in our
field experiment, as not all peaks corresponded to both indicators. By
providing time-integrated estimates of bacterial indicators in rivers,
the mussels monitored faecal pollution even 2 days after the peak expo-
sure. Such detection of faecal contamination would likely have failed
using conventional water grab sampling, as water concentrations
show a high temporal variability due to heavy precipitation leading to
wash off of pathogens from land and combined sewer overflows
(Tornevi et al., 2014) or pathogen resuspension from sediment
(Pachepsky and Shelton, 2011). Hence, biomonitoring using mussels is
a supplement to grab water sampling and provides a better identifica-
tion of hazards in the water bodies. While most studies on faecal path-
ogens in mussels address marine species of economical importance
(e.g., Mannas et al., 2014; Martinez-Urtaza et al., 2003), our study con-
tributes with new knowledge on a freshwater bivalve.
In our field study, water temperature and pH correlated with the
concentration of faecal indicators in different matrices. The observed
gradients in these environmental parameters were mainly driven by
the STP outlet. At this site, pH was one unit lower (7 instead of 8) and
temperature was 3 °C higher than at the other sites. Hence, we cannot
separate effects of pH and temperature, from those of the site, as these
variables co-vary. Also, rainfall and the concentration of faecal indica-
tors in either mussels or water did not correlate, likely due to low pre-
cipitation during our field experiment (average 0.073 mm in 24 h).
Depuration of cultured bacteria from mussels was incomplete. Mus-
sels still contained N40% of the initially accumulated bacterial indicators
after 60 h of depuration (Fig. S2), likely due to food deficiency and ces-
sation in filtering activity. In contrast, after 24 h of depuration from sew-
age effluent exposure, the indicator bacteria were basically
undetectable (Fig. 3). This discrepancy between the two experiments
might be due to the longer pulse duration and higher faecal indicator
concentrations used in the experiment with pure cultures (Table 1).
4.2. Antibiotic resistance
MDR bacteria in mussels exceeded concentrations in water by a fac-
tor of 2–5 in our experiments. This implies that sampling mussels is a
much more reliable approach than grab water samples for detecting re-
sistant microbes. Moreover, as the presence of resistant bacteria usually
is related to the occurrence of antibiotics in the environment (Levy and
Marshall, 2004), caged mussels can provide a time-integrated, low-cost
alternative for the detection of MDR in the environment. Furthermore,
the potential selective pressure for antibiotic resistance in the mussels
should be investigated in order to explain the higher predominance of
antibiotic resistant bacteria in mussel tissue than in ambient water. As
resistance can develop even at antibiotic concentrations below MIC,
mussels that contain only traces of antibiotics may potentially be reser-
voirs for resistance (Gullberg et al., 2011). In our study, we found a high
(4 out of 9) prevalence of antibiotic resistant Salmonella spp., but did not
observe any MDR isolates. Nonetheless, a recent report by the European
Food Safety Authority showed ‘extremely high’ MDR levels in some Sal-
monella serotypes (e.g. N70% MDR in S. infantis) (EFSA and ECDC, 2018).
We found a high frequency of resistance to ampicillin among E. coli iso-
lates from water in our field study, supporting the findings by Turolla
et al. (2018). In contrast, no resistant colonies to chloramphenicol
were found in our study.
Major sources of antibiotics to surface waters are runoff from farms
and landfills, as well as effluents from STPs. In our study, the STP outlet
was the main source of resistant bacteria, with similarly high propor-
tions of antibiotic resistant bacteria found at 1.5 km downstream. The
observed high prevalence of resistant bacteria in the river Fyrisån is of
serious concern for human health and underlines the need to prioritize
water safety management. Moreover, the high incidence of antibiotic
2148 M.A. Bighiu et al. / Science of the Total Environment 650 (2019) 2141–2149
Fig. 5. Proportion of antibiotic resistant and multidrug resistant (MDR) (A) E. coli and
(B) enterococci colonies isolated from mussels (solid bars) and water (striped bars),
sampled in both the field (left panels) and laboratory (right panels) experiments. Am =
ampicillin, Ci = ciprofloxacin, Em = erythromycin, Gm = gentamicin, Nal = nalidixic
acid, Sm = streptomycin, Vi = virginiamycin, Ba = bacitracin, Ctx = cefotaxime, Caz =
ceftazidime, Cm = chloramphenicol, Cs = colistin, Ff = florfenicol, Km = kanamycin,
Lz = linezolid, Na = narasin, Tc = tetracycline, Tm = trimethoprim, Va = vancomycin.
Note the different scales on the Y axes for graphs A and B.
resistant and MDR bacteria in mussels indicates a potential food safety
risk. For example, imported sea food has been identified as a source of
resistance genes in E. coli (Slettemeås et al., 2017) and Enterobacteria-
ceae (Janecko et al., 2016). Along with increasing awareness for the
human health risk, such observations are meaningful signals of the
dispersal and magnitude of resistant genes in aquatic ecosystems
(Baquero et al., 2008).
5. Conclusions and outlook
Our study shows the feasibility of zebra mussels as a time-
integrating alternative and/or supplement for biomonitoring faecal con-
tamination in inland waters, especially so for screening over large spa-
tial scales. Zebra mussels showed a rapid uptake of faecal indicators,
including antibiotic resistant ones and retained them for up to 2 days
after pulse exposure. This, in combination with their low susceptibility
to serious diseases (see above) and their wide distribution across
Europe and the US, make these mussels a suitable tool for the assess-
ment of sanitary water quality. Obviously, considering the invasiveness
of zebra mussels, biomonitoring should be restricted to areas where
they already are established. This limitation may be overcome in a
near future when males and females can be distinguished by new tech-
niques, thus allowing for the use of male-only populations. Although the
preparation of mussel samples for bacterial analysis may be more
labour-intensive than that for water samples, our study shows that a
sampling regime of 48–72 h increases the detection of antibiotic resis-
tant bacteria.
Acknowledgements
The authors thank Lise-Lotte Fernström for help with the laboratory
work, Stefan Hellgren for creating the map, and Björn Bengtsson for ad-
vice in interpreting the VetMic™ results. This study was funded by the
Non-Toxic Environment Programme of the Swedish University of Agri-
cultural Sciences (SLU).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.09.314.
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