Science of the Total Environment 408 (2009) 365–372

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Science of the Total Environment

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Antimicrobial resistance of fecal bacteria in waters of the Seine river

watershed (France)
Pierre Servais ⁎, Julien Passerat
Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Campus de la Plaine, CP 221, Boulevard du Triomphe, B-1050 Bruxelles, Belgium

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

Article history: We studied the prevalences of antimicrobial resistance (AR) and multiple antimicrobial resistance (MAR)
Received 24 April 2009 among the fecal bacteria found in the rivers of a large watershed under strong anthropogenic pressures, the
Received in revised form 14 September 2009 Seine river watershed (France). Two groups of fecal indicator bacteria, Escherichia coli and intestinal
Accepted 24 September 2009
enterococci, were tested for their susceptibility to 16 and 10 antimicrobials respectively, using the disk
Available online 24 October 2009
diffusion method. We found that 42% of the 214 E. coli river isolates were AR (resistant to at least one
Keywords:
antimicrobial) and 35% were MAR (resistant to at least two antimicrobials). Among the 148 intestinal
Antimicrobial resistance enterococci isolates from rivers, 83% were AR and 49% were MAR. We also investigated the sources of AR
Escherichia coli fecal bacteria found in the rivers of the watershed. A total of 715 E. coli isolates and 476 intestinal enterococci
Intestinal enterococci isolates were collected in point sources (municipal and hospital wastewaters) and non-point sources
River waters (surface runoff and soil leaching waters from agricultural or forest areas). For E. coli, the prevalence of AR
Wastewaters differed widely from source to source and ranked in this order: hospital wastewaters (71%) > municipal
wastewaters (44%) > agricultural non-point sources (16%) > forest non-point sources (2%). The prevalence of
MAR ranked similarly, and the same trend was observed for intestinal enterococci. The AR level of fecal
bacteria in the sources was related to their expected exposure level to antimicrobials before their release into
the environment. A MAR index was calculated for every source and a good discrimination between them was
thus obtained. At the global scale of the Seine river watershed, domestic wastewaters seemed more likely to
be the predominant source of the AR fecal bacteria found in the rivers. This was corroborated by the
similarity of the MAR indices from river and municipal wastewater isolates for both fecal indicators.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction posed by this resistance is so alarming that networks were developed

During the last fifty years the utilization of antimicrobial drugs has
steadily increased. Antimicrobials are extensively used in human
medicine to treat or prevent bacterial infectious diseases. In addition
to the large use for the treatment of human illnesses, antimicrobials
are commonly used in veterinary practices. Antimicrobials have also
been used for years at subtherapeutic concentrations as growth
promoters in animal husbandry, as it allows the increase of the animal
weight without increasing the food ration (Nwosu, 2001). This
practice is now strictly regulated in various countries and even totally
prohibited in the EU since 2006 (European Parliament and the
Council, 2003) but still continues in both the developed and
developing worlds. Indiscriminate use of antimicrobials has resulted
in the increase in the number of antimicrobial resistant (AR, resistant
to at least one antimicrobial) and multiple antimicrobial resistant
(MAR, resistant to at least two antimicrobials) bacteria. Today, the
presence of AR bacteria is a major public health concern. The problem
⁎ Corresponding author. Tel.: +32 2 6505995; fax: +32 2 6505993.
E-mail address: pservais@ulb.ac.be (P. Servais).
to monitor the antimicrobial resistance (AR) as the European
Antimicrobial Resistance Surveillance System (EARSS) and the
National Antimicrobial Resistance Monitoring System (NARMS) in
the United States. However, these networks usually focus only on AR
in human clinical isolates. Traditionally, the presence of AR bacteria in
aquatic systems has received much less attention.
Enteric bacteria from human and animal feces can be found in
surface waters; the fecal bacteria are brought into aquatic environ-
ments mainly through treated or untreated wastewater release,
surface runoff and soil leaching. The presence of pathogenic enteric
micro-organisms in aquatic environments can be a source of disease
when water is used for drinking water production, recreational
activities, shellfish harvesting or irrigation. The sanitary risk is of
course increased if the pathogenic enteric bacteria present in waters
are AR because human infections caused by such bacteria could be
difficult to treat with drugs. In addition, fecal bacteria might be able to
transmit AR to autochthonous bacteria through lateral transfer, when
the resistances genes are carried by transferable and mobile genetic
elements, thus contributing to the spread of AR.
A limited number of studies have investigated the presence of AR
fecal bacteria in waters. However AR fecal bacteria have been already

0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2009.09.042
366 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372
isolated from different types of surface waters: rivers (Boon and
Cattanach, 1999; Ash et al., 2002; Watkinson et al., 2007; Hamelin
et al., 2007), estuaries (Parveen et al., 1997), lakes (Edge and Hill,
2005) and coastal waters (Kimiran-Erdem et al., 2007). Studies also
reported a high proportion of AR fecal bacteria (Escherichia coli,
intestinal enterococci) in influents and effluents of wastewater
treatment plants (Reinthaler et al., 2003; Gallert et al., 2005; da
Costa et al., 2006; Boczek et al., 2007; Jakobsen et al., 2008). Several
studies have used the AR pattern of fecal bacteria to investigate the
origin of fecal pollution in various aquatic environments in an
approach called “microbial source tracking” (Wiggins et al., 1999;
Hagedorn et al., 1999; Whitlock et al., 2002; Harwood et al., 2000,
2003; Webster et al., 2004; Moore et al., 2005).
The first objective of this study was to evaluate the presence of AR
fecal bacteria in waters of the Seine river watershed (France). This
watershed (75,000 km2) located in the Northern part of France is
highly anthropogenically impacted, due to the high population den-
sity, intense industrial activities and intensive agriculture (Meybeck
et al., 1988). The large rivers of the Seine drainage network present a
quite high level of fecal contamination, especially in the highly
inhabited Parisian area (Servais et al., 2007). The second objective
of this study was to characterize the AR of fecal bacteria in micro-
bial pollution sources in order to determine the main origins of AR
fecal bacteria found in the rivers of the Seine drainage network.
The following sources were analyzed: (i) municipal and (ii) hospital
wastewaters in which fecal bacteria mainly originate from human
feces; (iii) surface runoff and soil leaching waters from agricultural
areas in which fecal bacteria mainly originate from grazing livestock
feces and manure spreading; (iv) surface runoff and soil leaching
waters from forest areas in which fecal bacteria may predominantly
originate from wild animal feces.
In this study, AR was investigated in two bacterial groups, E. coli
and intestinal enterococci, which are widely used as indicators of fecal
contamination of waters in the EU and throughout much of the world.
Fig. 1. Map of the Seine river watershed with location of the sampling stations in the rivers (white circles) and of the area in which the small streams were investigated (grey area).
E. coli and intestinal enterococci were enumerated and isolated on
specific agar media and the resistance of the isolates to antimicrobials
was evaluated using the disk diffusion method. Resistance of the
E. coli and intestinal enterococci isolates was tested against 16 and 10
antimicrobials respectively.
2. Materials and methods
2.1. Sample collection
All water samples were collected between March 2005 and
November 2007 in the Seine river watershed. Grab water samples
were collected, kept at 4 °C in sterile 1-litre bottles and analyzed
within 24 h.
River samples were collected in 9 rivers of the Seine river hydro-
graphical network, at 11 locations (Fig. 1). Each location was visited in
dry weather conditions one to four times during the course of the
study. The diversity of the sampled rivers encompassed weakly con-
taminated rivers up to the highly contaminated main rivers of the
watershed (Seine, Marne and Oise).
Municipal wastewater samples were collected in raw and treated
wastewater at the inlet and the outlet of 7 municipal wastewater
treatment plants (WWTPs). Samples were collected in each WWTP
one or two times during the course of the study. The nominal capacity
of the investigated WWTPs ranged from 8400 to 6,500,000 inhabi-
tant-equivalents. The treatment processes in these plants include
pretreatment (screening, grease collection), settling and different
biological treatments (activated sludge with or without nitrification
and denitrification). None of the treated wastewaters analyzed were
disinfected.
Hospital wastewater was sampled in the raw effluents from several
large hospitals located in the Seine watershed. Samples were collected
in the hospital sewer network before the raw hospital wastewaters
reach the municipal sewer network.
 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372
To collect samples of water resulting from the surface runoff and
soil leaching in agricultural and forest areas, small rural streams were
sampled upstream from any wastewater outfall to prevent their
contamination by point sources, and at a maximum of 2 km from their
source. Streams were characterized according to the main land use
(more than 75% of the surface of the watershed as determined by a GIS
approach) within their watershed. Eleven streams with a watershed
mainly covered by crops and pastures and 12 streams with a
watershed covered mainly by forest were sampled. These samples
are thereafter referred as agricultural non-point sources and forest
non-point sources respectively.
2.2. Enumeration and isolation of E. coli and intestinal enterococci
Abundance of the two fecal indicators in the water samples was
estimated by the membrane filtration method or by direct plating,
according to the presumptive contamination level. Chromocult
Coliform agar and Chromocult Enterococci agar (Merck KGaA,
Darmstadt, Germany) were used to enumerate E. coli and intestinal
enterococci, respectively. Plates were incubated at 36 °C for 24 h
(E. coli) or for 36 h to 48 h (intestinal enterococci). For E. coli, an
average of 10 typical colonies (dark-blue to violet colonies resulting
from the cleavage of Salmon-GAL and X-glucuronide by β-D-
galactosidase and β-D-glucuronidase respectively) per water sample
were randomly selected and isolated by streaking onto a new
Chromocult Coliform agar plate. The presumptive E. coli isolates
were further tested for tryptophanase activity with Kovac's reagent.
Only the isolates that were positive for the three enzyme activities
were considered as E. coli. For intestinal enterococci, for which
isolation was begun later in the course of the study, the isolation
effort was raised to an average of 16 typical colonies randomly
selected per water sample and streaked onto a new plate. Thirty five
percent of the presumptive isolates were further streaked onto bile
aesculin azide agar and all of them developed the characteristic
morphology and color of enterococci colonies. The presumptive
intestinal enterococci isolates selected on Chromocult Enterococci
agar were therefore tentatively considered as intestinal enterococci.
2.3. Antimicrobial susceptibility testing
Antimicrobial resistance was assessed for both fecal indicators
using the disk diffusion method, according to the recommendations
of the CA-SFM (Comité de l'Antibiogramme de la Société Française de
Microbiologie, 2005). Briefly, overnight cultures in Mueller–Hinton
broth (Merck KGaA, Darmstadt, Germany) were used to prepare a
bacterial suspension adjusted to a 0.5 McFarland standard. A 100-fold
dilution of the suspension was spread on Mueller–Hinton agar (Merck
KGaA, Darmstadt, Germany) with a sterile cotton swab. The antimi-
crobial disks (Bio-Rad, Marnes-la-Coquette, France) were dispensed
with automatic disk dispensers (6 disks per plate) and plates were
incubated for 24 h at 36 °C. The resistance of the E. coli and intestinal
enterococci isolates was tested against 16 and 10 antimicrobials res-
pectively. The tested antimicrobials belonged to the CA-SFM standard
and complementary lists of antimicrobials to test with Enterobacter-
iaceae and Enterococcus spp. respectively. They were chosen so that
the main classes of antimicrobials were represented in this study. The
antimicrobial content of the disks was that recommended by the
CA-SFM. The resistance of the E. coli isolates was tested against the
following antimicrobials: amoxicillin (AMX, content of the disk:
25 µg), amoxicillin/clavulanic acid (AMC, 20/10 µg), cefalotin (CF, 30 µg),
cefotaxime (CTX, 30 µg), ceftiotur (XNL, 30 µg), ceftazidime (CAZ, 30 µg),
tetracycline (TE, 30 µg), sulfamethoxazole/trimethoprim (SXT, 23.75/
1.25 µg), nalidixic acid (NA, 30 µg), ciprofloxacin (CIP, 5 µg), enroflox-
acin (ENR, 5 µg), levofloxacin (LVX, 5 µg), ofloxacin (OFX, 5 µg),
norfloxacin (NOR, 5 µg), gentamicin (GM, 15 µg) and amikacin (AN,
30 µg).
367
The resistance of the intestinal enterococci isolates was tested
against the following antimicrobials: sulfamethoxazole/trimethoprim
(SXT, 23.75/1.25 µg), erythromycin (E, 15 IU), levofloxacin (LVX,
5 µg), tetracycline (TE, 30 µg), ampicillin (AM, 10 µg), chloramphen-
icol (C, 30 µg), linezolid (LZD, 30 µg), gentamicin (GEN, 500 µg),
vancomycin (VA, 30 µg) and teicoplanin (TEC, 30 µg).
The reference strains E. coli ATCC 25922 and Enterococcus faecalis
ATCC 29212 were included as controls in each set of experiments. The
isolates were scored as susceptible, intermediate or resistant to a
given antimicrobial by the inhibition zone diameter around the disk
and according to the CA-SFM recommendations (CA-SFM, 2005).
Intermediate and resistant isolates were subsequently grouped in a
same resistant class, following Reinthaler et al. (2003).
The susceptibility testing of E. coli to XNL, LVX, CAZ, OFX and NOR,
and of intestinal enterococci in general, were only begun in the course
of the study. Therefore, in both cases, AR was investigated on fewer
samples and isolates (Table 1).
2.4. Data analysis
An isolate was considered AR if it was resistant to at least one of
the antimicrobials tested and MAR if it was resistant to at least two.
The prevalences of AR and MAR among a group of isolates were
therefore the percentages of AR and MAR isolates in this group. For
both fecal indicators, a MAR index was calculated for the four sources
of fecal contamination and for the rivers, as described in Krumperman
(1983). The MAR index is the proportion of negative susceptibility
tests (sum over all isolates of the number of antimicrobials to which
each isolate is resistant) in the total number of susceptibility tests
(number of antimicrobials tested multiplied by the number of
isolates). For E. coli, MAR indices were calculated by taking into
account only the results for the 11 antimicrobials that were tested
since the beginning of the study. Prevalences (of AR, of MAR and of the
resistance to a given antimicrobial) were compared between the
different water types and the differences were analyzed using a two-
sided chi-square test.
3. Results
3.1. Abundance of the fecal indicators in the studied samples
Abundances of the two fecal indicators found in the different water
samples collected in this study ranged over 6 logs, from forest non-
point sources to raw wastewaters (Table 2). The range of the
abundances found in rivers extended over 2.2 logs for E. coli and 2.4
logs for intestinal enterococci. The less contaminated rivers had
abundances similar to forest non-point sources and the most
contaminated rivers abundances similar to treated wastewaters. The
levels of fecal contamination of municipal wastewaters, agricultural
and forest non-point sources found in this study were in the same
order of magnitude than those reported by previous studies on the
microbial contamination in the Seine watershed (Garcia-Armisen and
Servais, 2007; Servais et al., 2007).
3.2. Antimicrobial resistance of E. coli and intestinal enterococci in river
samples
Among the 214 E. coli isolates recovered from river samples
(Table 1), 42% were resistant to at least one of the 16 antimicrobials
tested (Table 3). Resistances were principally observed against 5
antimicrobials: amoxicillin (33%), tetracycline (27%), sulfamethox-
azole/trimethoprim (16%), amoxicillin/clavulanic acid (16%), and the
first-generation cephalosporin cefalotin (14%) (Fig. 2A). Resistances
to amoxicillin, tetracycline and sulfamethoxazole/trimethoprim were
responsible for 96% of the AR E. coli in rivers (data not shown). Half of
the amoxicillin-resistant isolates were also resistant when amoxicillin
368 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372

Table 1
Number of water samples and number of fecal indicator isolates tested for their resistance to antimicrobials.

Water sample E. coli Intestinal enterococci

AMX, AMC, CF, CTX, XNL LVX CAZ, OFX, NOR SXT, E, LVX, TE, AM, C,
TE, SXT, NA, CIP, ENR, LZD, GEN, VA, TEC
GM, AN

Samples Isolates Samples Isolates Samples Isolates Samples Isolates Samples Isolates

Rivers 21 214 18 186 12 118 9 88 9 148

Hospital wastewaters 17 235 17 216 13 139 13 139 6 122
Municipal wastewaters 16 179 11 119 6 69 5 57 6 90
Agricultural non-point sources 14 159 12 139 9 107 5 70 8 131
Forest non-point sources 15 142 12 124 10 110 7 80 10 133
Total 83 929 70 784 50 543 39 434 39 624

Table 2
Abundances of fecal indicators in the different water samples.

Water sample E. coli Intestinal enterococci

Number of Geometric mean Range Number of Geometric mean Range

samples samples
CFU/100 mL CFU/100 mL CFU/100 mL CFU/100 mL

Rivers 21 2.1 103 2.7 102–4.5 104 9 8.1 102 4.5 101–1.2 104
Hospital wastewaters 17 1.5 106 2.7 104–3.6 107 6 2.7 104 4.1 103–9.3 105
Municipal wastewaters 16 8.4 105 1.2 104–1.5 107 6 2.0 105 3.4 103–2.1 106
Raw 9 6.5 106 7.9 105–1.5 107 3 1.8 106 1.6 106–2.1 106
Treated 7 8.1 104 1.2 104–2.9 106 3 2.1 104 3.4 103–9.4 104
Agricultural non-point sources 14 1.8 103 1.8 102–1.6 104 8 2.3 102 2.6 101–4.0 103
Forest non-point sources 15 1.1 102 1.5 101–2.7 103 10 3.5 101 5.0 100–1.3 102

CFU: Colony forming units.

was associated to clavulanic acid, a beta-lactamase inhibitor. chloramphenicol were low (8%, 4% and 4%), and no resistance was
Resistance to nalidixic acid was low (4%). Resistances to the third- observed to gentamicin, vancomycin or teicoplanin in the experi-
generation cephalosporins (cefotaxime, ceftiotur and ceftazidime), to mental conditions used in the present study. Almost half of the
the fluoroquinolones (ciprofloxacin, enrofloxacin, levofloxacin, oflox- isolates were MAR (49%) (Table 3).
acin and norfloxacin) and to the aminoglycosides (gentamicin and
amikacin) were very low (<2%). No resistant isolates were found for 3.3. Antimicrobial resistance of E. coli and intestinal enterococci from
levofloxacin and amikacin. Finally, a third of the isolates (35%) were different sources
demonstrated MAR (Table 3).
As regards intestinal enterococci, 83% of the 148 river isolates were In addition to the river samples, isolates of the two fecal indicators
resistant to at least one of the 10 antimicrobials tested (Table 3). This were collected from municipal and hospital wastewaters, and from
higher prevalence of AR in comparison to E. coli was mainly explained leachates from agricultural and forest areas (agricultural and forest
by the high prevalence of the resistances to sulfamethoxazole/ non-point sources). For the E. coli isolates from municipal waste-
trimethoprim (65%) and erythromycin (49%) (Fig. 2B), which were waters, the prevalences of AR (44%) and MAR (34%) were very similar
sufficient to explain 91% of the AR intestinal enterococci in rivers (data (not statistically different) to those observed in river waters (42% and
not shown). Resistances to levofloxacin and tetracycline were 35% respectively) (Table 3). Indeed, for 10 antimicrobials out of the 16
intermediate (20% and 18%). Resistances to linezolid, ampicillin and tested, the prevalences of resistance were not statistically different to
those found in river waters (Fig. 3A). Nevertheless, resistances to the 6
quinolones (nalidixic acid and the 5 fluoroquinolones) were more
Table 3
prevalent in municipal wastewaters than in rivers samples (P ≤ 0.05).
Percentages of fecal indicator isolates resistant to at least one (AR isolates), two (MAR With regard to the hospital wastewaters, prevalences of AR (72%)
isolates) or five antimicrobials in the different water samples, and resulting MAR and MAR (65%) were higher than in rivers and municipal wastewaters
indices. (P ≤ 0.001). This high level of AR found in hospital wastewaters was also
Water sample E. coli Intestinal enterococci observed in the number of antimicrobials to which isolates were
resistant: 35% of the hospital wastewater isolates were resistant to at
≥1 ≥2 ≥5 MAR ≥1 ≥2 ≥5 MAR
(AR) (MAR) % index (AR) (MAR) % index least 5 antimicrobials, against 8% and 2% for municipal wastewater and
% % % % river isolates respectively (P ≤ 0.001) (Table 3). Resistance against every
Rivers 42 35 2 0.075 83 49 2 0.168
antimicrobial tested was found (Fig. 3B). For all antimicrobials, the
Hospital 71 65 35 0.250 89 66 15 0.240 prevalence of resistance was at least twice as high as that for municipal
wastewaters wastewaters. Resistances to quinolones were even three to four times
Municipal 44 34 8 0.093 78 53 6 0.174 more prevalent than in municipal wastewaters; resistance to gentami-
wastewaters
cin was seven times more prevalent. A low level of resistance to the
Agricultural 16 8 0 0.019 66 11 0 0.078
non-point third-generation cephalosporins was observed, while it was very low or
sources not observed in rivers and municipal wastewaters.
Forest 2 1 0 0.003 32 6 0 0.041 In agricultural and forest non-point sources, conversely, very low
non-point prevalences of AR and MAR were found (Table 3). In forest non-point
sources
sources, resistances to only five antimicrobials were detected, and
 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372 369

Fig. 2. Prevalences of resistance of E. coli (A) and intestinal enterococci (B) isolates from river samples to various antimicrobials.

their prevalence did not exceed 1% (Fig. 3D). The five antimicrobials
corresponded to those with the highest prevalences of resistance in
rivers. In agricultural non-point sources, the prevalences of resistance
to these antimicrobials were higher, but lower than 10% (Fig. 3C).
Resistance to nalidixic acid was also detected, with a prevalence of 1%.
For every other antimicrobial, no resistance was observed.
The four sources had very different MAR indices that increased in
this order: forest non-point sources (0.003), agricultural non-point
sources (0.019), municipal wastewaters (0.093) and hospital waste-
waters (0.250) (Table 3). Rivers had a MAR index close but lower than
municipal wastewaters (0.082).
For intestinal enterococci, the same trend was observed: the
prevalence of AR, the prevalence of MAR and MAR indices increased in
the same order from source to source (Table 3). For each antimicrobial
taken separately, the percentage of resistant isolates also increased
according to the trend, with the exception of linezolid and
sulfamethoxazole/trimethoprim (Fig. 4). For these two antimicrobials,
the prevalence of resistance was not statistically different in
agricultural non-point sources, in municipal and in hospital waste-
waters. The range of MAR indices between the sources was narrower
than observed with E. coli. This was mainly due to the higher MAR
index found in forest non-point sources, which resulted from an
intermediate level of resistance to sulfamethoxazole/trimethoprim
(24%) and erythromycin (11%). As for E. coli, the MAR index of river
isolates was close but slightly lower than the MAR index of municipal
wastewater isolates (0.168 and 0.174 respectively). Finally, no isolate
resistant to teicoplanin or vancomycin was found in this study.
3.4. Comparison of antimicrobial resistance of E. coli and
intestinal enterococci
In 38 samples, AR was tested on both E. coli and intestinal
enterococci and MAR indices were calculated. In Fig. 5, MAR indices of
intestinal enterococci were plotted against MAR indices of E. coli.
There was no statistically significant correlation between both indices
but, however, data showed a positive trend indicating that AR of
intestinal enterococci tended to increase when AR of E. coli increased.
4. Discussion
In this study, 42% of the E. coli found in the rivers of the Seine
watershed were AR, and 35% were MAR. As regards intestinal
enterococci, the prevalences of AR and MAR were of 83% and 49%
respectively. The four sources of fecal bacteria investigated in this

Fig. 3. Prevalences of resistance of E. coli isolates from municipal wastewaters (A), hospital wastewaters (B), agricultural non-point sources (C) and forest non-point sources (D) to
various antimicrobials.
370 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372
Fig. 4. Prevalences of resistance of intestinal enterococci isolates from municipal wastewaters (A), hospital wastewaters (B), agricultural non-point sources (C) and forest non-point
sources (D) to various antimicrobials.
study were very different in terms of AR and MAR prevalences. For
both fecal indicators, the prevalences of AR and MAR increased in this
order: non-point sources from forest areas, non-point sources from
agricultural areas, municipal wastewaters and hospital wastewaters.
Some papers already presented data on the AR of fecal bacteria in
rivers. Among these, Hu et al. (2008) reported a prevalence of AR
among E. coli very similar to the one found in this study (48%), but
Webster et al. (2004) reported 9% of AR E. coli in a rural river, while
Sayah et al. (2005) found 80.6% of AR E. coli in the drainage network of
a watershed dedicated to swine and dairy cattle breeding. Our study
shows that, depending on the main source of fecal contamination that
impacts the river, the prevalence of AR can vary greatly and therefore
these differences are not surprising. Moreover, we observed that
resistances to 3 out of the 16 antimicrobials we tested were
responsible for 96% of the AR E. coli found in rivers. This stresses
that the estimate of the prevalence of AR is dependent on the
antimicrobials tested in a given study, making comparisons between
studies difficult, if not impossible. Nevertheless, our finding that
resistances to penicillins, tetracyclines and sulfonamides are more
Fig. 5. Intestinal enterococci MAR index plotted against E. coli MAR index for the samples
in which AR of both indicators was tested (n = 38).
prevalent in E. coli than resistances to quinolones and aminoglyco-
sides was consistent with previous reports (Reinthaler et al., 2003;
Watkinson et al., 2007; Hu et al., 2008). This is in good agreement with
the extent to which these antimicrobial classes have been prescribed
to date. Penicillins, tetracyclines and sulfonamides have been
extensively used for decades, while introduction of quinolones (and
more particularly fluoroquinolones) and aminoglycosides into clinical
use is more recent (Davies, 2007). In France, penicillins and
tetracyclines are still the first and third most prescribed antimicrobial
classes in ambulatory care, with 52% and 12% of the total defined daily
doses (DDD) respectively, while quinolones and aminoglycosides
represent 8% and less than 2% respectively (ESAC, 2006). For intestinal
enterococci, prevalences of resistance to erythromycin and tetracy-
cline similar to the ones found in this study were observed in fecal
samples of healthy human or in municipal WWTPs (da Costa et al.,
2006; Poeta et al., 2006). A very high prevalence of resistance to
sulfamethoxazole/trimethoprim was also reported in dairy (90%) and
clinical isolates (77%)(Lopes et al., 2005). Finally, since they are a
cause of public health concern (Klare et al., 2003), it is important to
point out that no glycopeptide-resistant enterococci were found in
this study.
The four sources of fecal contamination characterized in this study
have been investigated because they differ in terms of the origin of the
fecal bacteria they release in the rivers (human, livestock or wildlife)
and in the expected exposure of these bacteria to antimicrobial
selective pressure. Indeed, the correlation between antimicrobial
consumption and AR of commensal bacteria is now well established
(van den Bogaard and Stobberingh, 2000), and one can assume that
the degree to which fecal bacteria are exposed to antimicrobials
before their release in the environment is the primary reason for the
levels of AR observed in the river samples. The observation that the
prevalences of AR and MAR increased from forest non-point sources
to hospital wastewaters for both fecal indicators was in accordance
with this hypothesis. Firstly, in forest non-point sources, fecal bacteria
are likely to have originated from wildlife and are therefore less likely
to have been exposed to antimicrobials. Secondly, the prevalences of
AR and MAR were higher in hospital wastewaters than in municipal
 P. Servais, J. Passerat / Science of the Total Environment 408 (2009) 365–372
wastewaters, a result consistent with the antimicrobial consumption
in human medicine in France. In 2006, the antibiotic uses in hospital
care (systemic use) and ambulatory care were respectively estimated
at 2.32 and 27.9 DDD per 1000 inhabitants per day (ESAC, 2006). On
the other hand, 136,000,000 complete hospitalization days were
totalized in 2004 (Ministère de la Santé et des Solidarités, 2006) for a
population of 60,200,000 inhabitants (Desplanques and Royer, 2005),
indicating that 0.6% of the French metropolitan population is
hospitalized daily. Therefore an antibiotic use of 387 DDD per 1000
inpatients and per day is a better estimate of the exposure of fecal
bacteria to antimicrobials in hospital care; it exceeds by one order of
magnitude the exposure in ambulatory care.
Additionally, the prevalences of AR and MAR were lower in
agricultural non-point sources than in municipal wastewaters. This
result seems also consistent with the data of antimicrobial consump-
tion in human and veterinary medicines in France. In 2005, the sales of
veterinary and human antimicrobials were respectively of 1320 and
760 tons. In order to compare both usages in terms of selective
pressure, Moulin et al. (2008) evaluated that they resulted in an
annual consumption of 84 and 199 mg of active components per
kilogram of live weight in animal and human populations respective-
ly. The antimicrobial consumption in veterinary medicine was
therefore supposed to exert on average a selective pressure lower
than in human medicine, but antimicrobial usages are very hetero-
geneous among the different livestock productions. Since (i) cattle are
the main livestock in our area of study and (ii) specific data for the
different livestock indicated that the mass of antimicrobials consumed
per live weight is much lower for cattle than for swine and poultry, we
could expect the value of 84 mg/kg to overestimate the real
antimicrobial exposure of the fecal bacteria found in the agricultural
non-point sources from the Seine watershed.
Thus, AR levels of fecal bacteria found in the different sources were
roughly consistent with the exposure level to antimicrobials that these
bacteria were expected to have experienced before their release into
the environment. This is in agreement with what was already observed
for fecal bacteria directly isolated from feces of humans and various
animals (Wiggins, 1996; Hagedorn et al., 1999). For both fecal
indicators, MAR index proved a useful tool to characterize the global
level of AR in fecal bacteria from the different water origins. It
discriminated well the different sources, as observed in previous
studies (Parveen et al., 1997; Guan et al., 2002). It was nevertheless
less discriminating for intestinal enterococci than for E. coli. First, the
range of MAR indices between the sources was narrower for intestinal
enterococci, due in part to the already high prevalences of resistance to
sulfamethoxazole/trimethoprim and erythromycin in the forest
samples not exposed to antimicrobials. This could be explained by
the fact that some enterococci strains are naturally resistant to
sulfonamides (CA-SFM, 2005). This result stresses the importance in
choosing the antimicrobials to test in order to establish a MAR index.
Second, contrarily to E. coli, the upward trend of the prevalence of AR
from forest non-point sources to hospital wastewaters was not
observed for all antimicrobials. Intestinal enterococci form a group of
species whose AR properties can vary notably. For example, the
prevalence of resistance to ampicillin is about 60–80% among E.
faecium strains and ≤2% among E. faecalis strains (Klare et al., 2003).
These species do not have the same prevalence in human or in animal
feces (Klein, 2003). Therefore this could result in intestinal enterococci
with a pattern of AR differing from source to source independently
from their exposure level to antimicrobials.
In a previous study, the respective contributions of point and non-
point sources of the fecal contamination of the rivers of the Seine
watershed were evaluated (Garcia-Armisen and Servais, 2007) and
showed that point sources were responsible at the least for 95% of the
E. coli found in the rivers. By combining this evaluation with the
present results of AR, it may be possible to determine the main sources
of AR fecal bacteria in rivers at the global scale of the watershed.
371
Because the prevalence of AR is much higher in point sources than in
non-point sources, the releases of wastewater seemed more likely to
be the major sources of AR fecal bacteria. The respective contributions
of domestic and hospital wastewaters in these releases are unknown.
They can be estimated by the proportion of the total population that is
hospitalized daily, as calculated above. It indicates that more than 99%
of the fecal bacteria in point sources are expected to have a domestic
origin. Therefore, even if the prevalence of AR among the E. coli from
hospital wastewaters is twice the prevalence found in domestic
wastewaters, the latter are by far the major contributor to the AR fecal
bacteria found in the rivers of the Seine watershed. Consistently, the
MAR indices of E. coli and intestinal enterococci isolates from rivers
were close to those found in municipal wastewaters. However,
concerning the spread of AR genes, the contribution of AR and MAR
fecal bacteria contained in hospital wastewaters could be predominant
due to high levels of resistance of these bacteria.
5. Conclusion
AR E. coli and intestinal enterococci were found at a high frequency
in the rivers of the Seine watershed. The majority of the AR isolates
were MAR. The level of AR varied broadly according to the source of
fecal bacteria. It was the highest in point sources investigated, first in
hospital wastewaters then in municipal wastewaters. It was the
lowest in non-point sources investigated, first from forest areas and
then from agricultural areas. These levels of AR were roughly
consistent with the exposure level to antimicrobials that fecal bacteria
were expected to have experienced before their release into the
environment. In our study, AR fecal bacteria found in rivers seemed
more likely to originate mainly from domestic wastewaters while
non-point sources of fecal bacteria contribute very little to the global
load of AR fecal bacteria in the rivers at the scale of the whole
watershed. Quantitatively, hospital wastewaters contributed far less
than domestic wastewaters to the discharge of AR fecal bacteria in the
rivers, but they were responsible for the dissemination into the
aquatic environment of strains with a much higher level of MAR.
Acknowledgements
This study was part of the PIREN-Seine research program of the
Centre National de la Recherche Scientifique (France). The authors
thank Sophie Reis and Stéphanie Duchateau for their laboratory works
on antibiotic resistance estimation. The authors thank Joëlle Eurin and
Fatima Tamtam for their help in the sample collection.
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