ARTICLE

Is Escherichia coli urinary tract infection a zoonosis?

Proof of direct link with production animals and meat
L. Jakobsen & P. Garneau & G. Bruant & J. Harel &
S. S. Olsen & L. J. Porsbo & A. M. Hammerum &
N. Frimodt-Mller
Received: 17 June 2011 / Accepted: 3 September 2011
# Springer-Verlag 2011
Abstract Recently, it has been suggested that the Escherichia
coli causing urinary tract infection (UTI) may come from
meat and animals. The purpose was to investigate if a clonal
link existed between E. coli from animals, meat and UTI
patients. Twenty-two geographically and temporally matched
B2 E. coli from UTI patients, community-dwelling humans,
broiler chicken meat, pork, and broiler chicken, previously
identified to exhibit eight virulence genotypes by microarray-
detection of approximately 300 genes, were investigated for
clonal relatedness by PFGE. Nine isolates were selected and
tested for in vivo virulence in the mouse model of ascending
UTI. UTI and community-dwelling human strains were
closely clonally related to meat strains. Several human
derived strains were also clonally interrelated. All nine
isolates regardless of origin were virulent in the UTI model
with positive urine, bladder and kidney cultures. Further,
isolates with the same gene profile also yielded similar
bacterial counts in urine, bladder and kidneys. This study
showed a clonal link between E. coli from meat and humans,
providing solid evidence that UTI is zoonosis. The close
relationship between community-dwelling human and UTI
isolates may indicate a point source spread, e.g. through
contaminated meat.
Introduction
Escherichia coli exists as a common commensal in the
intestines of humans and mammals. Apart from various
toxin-related diarrheal infections E. coli also causes extra-
intestinal infections, including urinary tract infections
(UTI). The disease burden of UTI is enormous. UTI is
believed to be the most common bacterial infection with an
estimated 150 million cases globally [1, 2]. In addition, a
considerable number of E. coli UTI cases results in
bacteremia and consequently deaths. It has been estimated
that one of every three women will experience a case of
treatment-requiring UTI before the age of 26 and 4050%
of women will experience at least one UTI during their
lifetime [3]. About 2530% of, otherwise healthy, women
with UTI will develop a recurrent infection [4]. In the U.S.
alone, the estimated cost burden is approaching 1 billion
dollars yearly [5].
Extraintestinal pathogenic E. coli, designated ExPEC, is
responsible for 8090% of the UTI cases [1, 4]. Most often
the E. coli belongs to phylogroup B2, characterized by
possessing numerous virulence genes allowing the isolate
to cause infection outside the intestinal tract, e.g. by
L. Jakobsen (*)
:
S. S. Olsen
:
A. M. Hammerum
:
N. Frimodt-Mller
Department of Microbiological Surveillance and Research,
Statens Serum Institut,
Build. 47/213, restads Boulevard 5,
2300 Copenhagen S, Denmark
e-mail: lja@ssi.dk
L. J. Porsbo
National Food Institute, Technical University of Denmark,
Copenhagen, Denmark
P. Garneau
:
J. Harel
Groupe de Recherche sur les Maladies Infectieuses du Porcine,
Facult de mdecine vtrinaire, Universit du Montreal,
Montreal, QC, Canada
G. Bruant
Biotechnology Research Institute,
National Research Council of Canada,
Montreal, QC, Canada
N. Frimodt-Mller
Department of Clinical Microbiology, Hvidovre Hospital,
30 Kettegrd alle,
DK-2650 Copenhagen, Hvidovre, Denmark
Eur J Clin Microbiol Infect Dis
DOI 10.1007/s10096-011-1417-5
adhering to host surfaces, scavenge iron or other micro-
nutrients, or evade host response [6, 7]. UTI is most often
caused by enteric E. coli. This transmission route is widely
referred to as the feacal-perineal-urethral route [8, 9]. It has
been suggested that the exterior source of ExPEC to the
intestine may be food and animals [10]. However, studies
that link UTI isolates with food and animal isolates due to
clonal relatedness using highly discriminative typing
methods, like pulsed-field gel electrophoresis, are lacking.
In this study, we investigate the clonal relatedness of
geographically and temporally matched E. coli from UTI
patients, community-dwelling humans, broiler chicken
meat, broiler chickens, pork, and pigs as well as the
virulence of non-UTI isolates in a mouse UTI model which
is representative of human UTI [11].
Materials and methods
Bacterial strains
A strain collection at Statens Serum Institut previously
described with respect to phylogroups and virulence
genes were used for this study [12, 13]. In short, E. coli
isolates were collected in Denmark in 2004 from
community-dwelling humans (n=102), Danish (n=197)
and imported broiler chicken meat (n=86), broiler chickens
(n=138), Danish (n=177) and imported pork (n=10), and
pigs (n=145) by using a stratified sampling scheme as part of
the Danish Integrated Antimicrobial Resistance Monitoring
and Research Programme (DANMAP) [14]. Community-
dwelling humans were chosen by a selection algorithm so
that they represented the age and gender distribution of the
total Danish population taking the differential participation
rates of various demographic groups into account (scientific
ethical committee approval (KF) 01-006/02). A total of 102
UTI isolates were collected later from November 2005 to
April 2006 in a general practitioner clinic (serving approx-
imately 10,800 persons) south of Copenhagen from patients
with community-acquired uncomplicated or complicated
UTI [12]. UTI isolates were deliberately sampled later than
animal, meat, and community-dwelling human isolates to
allow time for consumption of the meat and possible
colonization and invasiveness of the E. coli isolates.
Previously, isolates belonging to phylogroup B2 were
identified among all origins and 161 B2 isolates were
investigated for more than 300 virulence genes and 80
resistance genes using a microarray [12, 13]. A number of
strains within and across isolate origins shared identical gene
profiles and was used for this study [13]. We analyzed a total
of 22 B2 E. coli strains from UTI patients (n=10),
community-dwelling humans (n=6), Danish (n=3) and
imported broiler chicken meat (n=1), broiler chickens
(n=1), and Danish pork (n=1). These 22 isolates exhibited
eight different virulence gene profiles according to the
microarray data (Table 1). The eight gene profiles
represented either (i) isolates from several origins, i.e.
both animal and meat origin, or meat and human origin, or
from both human origins (gene profile 15) or (ii) they
consisted of UTI isolates only (gene profile 68) (Table 1).
All 22 isolates, based on their microarray virulence gene
profile, were previously designated to belong to the
pathotype ExPEC [13]. The E. coli species identity was
confirmed using API20E (Biomrieux, France) according
to the manufacturers instructions. Dietary habits, contact
with animals, travel and occupation of the community-
dwelling humans were obtained by questionnaire. Address
and family names were obtained for UTI patients where
possible.
Pulsed-field gel electrophoresis
To investigate the clonal relatedness between isolates of the
same gene profile, we subjected the 22 strains to pulsed-
field gel electrophoresis (PFGE) with XbaI using the
protocol made available by CDCs PulseNet [15, 16].
Salmonella Braenderup H9812 was used as molecular size
standard [16]. All strains were processed on one gel. The
PFGE profiles were analyzed using the Bionumerics
software, version 6.01 (Applied Maths, Sint-Martens-
Latem, Belgium) resulting in a dendrogram (1% tolerance,
1% optimization). The PFGE patterns were interpreted
according to Tenover et al. [17]. Accordingly, PFGE
profiles with four to six bands difference were considered
to be possibly related and profiles with two to three bands
difference were considered closely related.
Mouse model of ascending urinary tract infection
To investigate virulence of the non-UTI isolates, nine
strains from community-dwelling humans, Danish and
imported broiler chicken meat, broiler chickens, and Danish
pork, selected among isolates with gene profiles 15, were
tested in a mouse model of ascending UTI (animal
inspectorate approval no. 2004/561-835). The E. coli O
rough:H- D1923 was used as negative control in the mouse
experiments. The mouse model has been described previ-
ously [18, 19]. In short, mice bladders were emptied by
gently pressing their abdomen and 50 l (10
8
CFU) of each
bacterial suspension was inoculated slowly transurethrally
in six outbred female albino OF1 mice (2832 gram,
Charles River Laboratories, France) by use of plastic
catheters. Ninety-six hours post inoculation urine was
collected from each mouse by carefully pressing the
abdomen. The mice were then euthanized by cervical
dislocation, and bladder and kidneys were removed and
Eur J Clin Microbiol Infect Dis
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Eur J Clin Microbiol Infect Dis
stored in Eppendorf tubes. All urine samples were
processed the same day by spotting (20 l) of a series of
tenfold dilutions (10
0
10
-6
) in duplicates on bromthymol
blue agar plates (SSI Diagnostika). The bladder and
kidneys were stored in 0.9% saline solution and kept
at 80C until processing. They were then incubated at
room temperature for 1 h and subsequently homogenized
using a TissueLyser (Qiagen, Ballerup, Denmark). Plates for
bacterial counting were processed as described above.
The detection limit was 25 CFU/ml. Bacterial counts for
the different treatment groups were compared using the
Mann-Whitney test (two-tailed) with a significance level
of p0.05 (GraphPad Prism 5, GraphPad Software, San
Diego, CA, USA).
Results
Bacterial strains
A total of 22 phylogroup B2 E. coli isolated from UTI
patients, community-dwelling humans, Danish and
imported broiler chicken meat, broiler chickens, and Danish
pork were analyzed. Gene profile overview, isolation date,
number of virulence genes, gender and age for human
isolates are summarized in Table 2. Two strains were
obtained from a gardener (isolate 2030c04, gene profile 3)
and a house wife (isolate 2174c04, gene profile 4) who
reported eating pork and broiler chicken on a regular basis,
and having been in contact with dogs and cats but not with
food-producing animals over a 7-day period prior to the
sampling date, and had not travelled within the last three
months. The gardener further reported being in contact with
captive frogs and the house wife had been in contact with
wild rabbits. Isolate 2571c04 (gene profile 5) was obtained
from a 5-year-old kindergarten pupil. He was eating pork
and broiler chicken on a regular basis, had been in contact
with dogs and cats (but not production animals) over a 7-day
period prior to the sampling date, and had been on a 5-day trip
to Sweden within the last three months. The other three
community-dwelling humans (all women, aged 3 (2180c04),
44 (2235c04), and 60 (2168c04), respectively) did not
complete the questionnaire. None of the UTI patients shared
address or family name which indicated that they seemingly
may be unrelated.
PFGE typing
In the absence of alternative analysis methods, we used
Tenovers interpretation criteria in our study, which were
not intended for independently collected isolates from
humans, meat, and animals [17]. The 22 isolates yielded
13 PFGE types of closely related (n=11), possibly related
(n=5), or unrelated isolates (n=6) (Table 2). Only isolates
with identical virulence gene profiles were possibly or
closely related and no isolates from different gene profiles
shared related PFGE profiles. Two broiler chicken meat
isolates were closely related to a UTI isolate (a 3-band
difference, 92% similarity, gene profile 2) and a pork isolate
was closely related to a community-dwelling human isolate
(a 2-band difference, 96% similarity, gene profile 3) (Fig. 1).
Virulence in mouse UTI model
The bacterial counts in urine, bladder, and kidneys of the
nine strains from animals, meat and community-dwelling
humans are shown in Fig. 2. Results show that all nine
isolates had positive urine, bladder, and kidney cultures
ranging from 10
4
to 10
7
CFU in the urine, 10
3
10
6
CFU in
the bladder and 10
2
10
5
CFU in the kidneys (Fig. 2).
Isolates with identical gene profiles mostly (profiles 1, 3,
and 4) had statistically similar bacterial counts. Isolates
763301910 and 2235c04 (gene profile 2) had similar
bacterial kidney counts, but not urine (p 0.037) or bladder
(p 0.026) counts. The negative control strain failed to
produce any bacterial counts above the detection limit
except for one positive bladder culture.
Discussion
Outbreaks of community-acquired UTI have been reported
in Europe and Northern America, and closely related or
indistinguishable UTI isolates from unrelated women have
suggested point source dissemination [2024]. Although
transmission routes were not investigated food may have
been a possible source, as an epidemiological study has
previously suggested that frequent pork and chicken
consumption were related to multidrug-resistant UTI [25].
Similarity between animal, food, and human ExPEC
isolates with respect to phenotypic antimicrobial resistance
and virulence genotypes, and other molecular character-
istics have been demonstrated by several researchers,
including us, supporting the hypothesis that UTI may be a
zoonosis [12, 13, 2631]. This was substantiated recently
by the first study demonstrating virulence of E. coli isolates
from healthy production animals and fresh retail meat in a
mouse model of ascending UTI [19]. However, studies
providing evidence of a clonal link between UTI and
animals and meat E. coli using a highly discriminative
method like PFGE are few. Ramchandani et al. investigated
if E. coli strains belonging to clonal group A (CGA) could
be traced to food animals and identified one E. coli strain
from a cow isolated in 1988 with a 94% similar PFGE
pattern to a human CGA isolate from 1999 [10]. However,
no epidemiological connection was known, neither did the
Eur J Clin Microbiol Infect Dis
isolates share antibiotic susceptibility patterns nor virulence
gene profiles. Still, PFGE is a highly discriminating method
and with the high diversity within the E. coli species,
finding indistinguishable or related isolates are remarkable
[10]. However, the pathogenecity of the cow isolate was not
investigated and thus the zoonotic potential is unknown.
Another study by Johnson et al. demonstrated two PFGE
profiles with a 3-band difference of two faecal E. coli
isolates from a chicken (from 1997) and a human volunteer
(sampled during 19961998) [32]. Both isolates were 076
antigen positive and contained fimH, iutA, traT, and ompT
when screened for 35 virulence genes and 13 papA alleles.
Even though no evident epidemiological connection was
shown, the strength of the study by Johnson et al. was the
analysis of isolates from the same locale and the 3-year-
sampling period as compared to the study by Ramchandani
et al. Like in the study by Ramchandani et al. the
pathogenecity of the chicken isolateand thus the zoonotic
potentialalso remained uninvestigated [10, 32]. Finally,
recent studies reported clonal connections between a retail
Table 2 Background information on E. coli B2 strains from different origins sharing identical microarray virulence gene profile
Strain Isolate origin
a
Isolate obtained
(day-month-year)
PFGE
type
b
Virulence genes
shared
Background
information
Gender Age
(years)
Gene profile 1
7630253-1 Broiler chicken (feces) 07-04-2004 A
a
39
7633034-2 Danish broiler chicken meat 16-04-2004 A
a

7633037-4 Imported broiler chicken meat 23-04-2004 B
Gene profile 2
7633004-8 Danish broiler chicken meat 21-01-2004 C
a
47
7633019-10 Danish broiler chicken meat 20-02-2004 C
a

2235c04 Comm. humans (feces) 21-10-2004 D F 44
357-a UTI patient (comp., recurrent) 29-03-2006 C
a
F 62
Gene profile 3
7633121-6 Danish pork 04-10-2004 E
a
47
2030c04 Comm. humans (feces) 28-10-2004 E
a
M 47
Gene profile 4
2168c04 Comm. humans (feces) 21-10-2004 F 47 F 60
2174c04 Comm. humans (feces) 28-10-2004 G
b
F 56
2180c04 Comm. humans (feces) 21-10-2004 G
b
F 3
328-a UTI patient (uncomp.) 22-03-2006 G
b
F 38
Gene profile 5
2571c04 Comm. humans (feces) 14-01-2005 H
a
51 M 5
181-a UTI patient (uncomp.) 01-02-2006 H
a
F 28
Gene profile 6
69-a UTI patient (comp., elderly) 04-01-2006 I 38 F 80
72-a UTI patient (uncomp.) 04-01-2006 J F 38
Gene profile 7
199-a UTI patient (uncomp.) 09-02-2006 K
b
52 F 49
265-a UTI patient (comp., elderly) 09-03-2006 K
b
F 88
268-a UTI patient (comp., recurrent) 06-03-2006 L F 45
Gene profile 8
278-a UTI patient (comp., elderly) 08-03-2006 M
a
46 F 78
299-a UTI patient (comp., pregnancy) 16-03-2006 M
a
F 27
Comm. humans community-dwelling humans; Comp. complicated UTI due to (i) recurrent, recurrent case of UTI, (ii) elderly, elderly UTI patient,
or (iii) pregnancy, pregnant UTI patient; Uncomp. uncomplicated UTI
a
Closely related isolates (23 PFGE bands difference)
b
Possibly related isolates (46 PFGE bands difference)
Eur J Clin Microbiol Infect Dis
chicken and UTI isolates, and between APEC isolates and
human ExPEC isolates (from asymptomatic bacteruria,
cystitis, urosepsis, intestinal sepsis, and respiratory sepsis)
[33, 34].
In our study, we selected 22 B2 isolates with eight
different gene profiles identified previously by microarray
analysis of more than 300 virulence genes. With such an
extensive array of traits finding isolates with identical gene
profiles sharing 3647 genes was very unlikely even
though the isolates were geographically and temporally
matched. The identical gene profiles in themselves suggest
a connection between the isolates [13]. We therefore
investigated the PFGE profiles to explore any clonal
relatedness. This study revealed the association of (i) two
broiler chicken isolates and one UTI isolate with closely
clonally related PFGE profiles showing a 3-band difference
and (ii) one pork isolate and one community-dwelling
human isolate with closely related PFGE profiles (2-band
difference). This finding provides evidence of a clonal link
between E. coli from meat, humans and UTI thus
suggesting that UTI is a zoonosis. Although we did not
investigate directionality of the transmission but a possible
link exclusively, it is much more likely that the UTI patient
acquired the isolate from animals through the food chain
than a UTI patient transmitting isolates to food and animals,
hence the term zoonosis. We also identified closely and
possibly clonally related isolates from community-dwelling
humans and UTI patients as well as from UTI patients only.
To our knowledge, these isolates were epidemiologically
unrelated. Clonally related UTI isolates from otherwise
unrelated UTI patients have been demonstrated in studies
which suggested, based on previous investigations, that it
could be related to a point-source spread of these E. coli
from, e.g., pork and chicken [25, 35]. This could also be the
case in this study as the meat products in Denmark are
distributed nationwide and the community-dwelling
humans, who completed the questionnaire, all reported
eating chicken and pork several times a week. Unfortu-
nately, no dietary information could be collected from the
UTI patients. Further, as mentioned previously, outbreaks
of community-acquired UTI have been reported previously
which substantiates the theory of point source spread [20
23]. From a food safety perspective, foodborne UTI isolates
are of great concern as both broiler chicken meat and pork
are frequently consumed many places. In comparison,
fewer people are exposed to livestock why the overall risk
of acquiring UTI isolates through direct contact with
animals is lower.
Some isolates with identical gene profiles did not have
clonally related PFGE profiles, e.g. isolates with gene
profile 1, 2, and 4. Obviously, this may be due to the fact
that the strains are unrelated. It may also be explained by
the time elapsed between the sampling of isolates allowing
for the genome to change (e.g. gene profiles 2 and 4). Due
to the plasticity of the E. coli genome, differences may
likely arise on a PFGE profile. Further, we have no
knowledge of which isolate is the original isolate (outbreak
isolate) or if it is present among the isolates in our
collection. We may therefore be comparing isolates not to
the parental strain but to other diverted isolates.
A second finding is the demonstration of in vivo
virulence of E. coli isolates from a broiler chicken, meat
and community-dwelling humans in the mouse model of
ascending UTI. The mouse model is representative of
human UTI and is thus a very important tool for
investigating the hypothesis of UTI being a zoonosis,
which molecular characterization cannot replace [11]. This
further supported a previous study, where we demonstrated
the virulence of B2 animal and meat isolates in the mouse
UTI model [19]. Interestingly, isolates with identical gene
profiles had also identical in vivo virulence profiles. This
was consistent for isolates tested in vivo from gene profile
1, 3, and 4 even though isolates from gene profiles 1 and 4
had different PFGE types, respectively. This clearly
demonstrated that meat isolates are similar to human
isolates and that isolates from a broiler chicken, fresh meat
as well as community-dwelling humans can cause UTI.
Taken together, the above findings provide solid typing and
in vivo evidence that UTI is at times a zoonosis.
Limitations of the study include the limited knowledge
of epidemiological information on UTI patients, e.g. dietary
habits, as well as uncompleted questionnaires from three of
Fig. 1 PFGE profiles of
selected E. coli B2 isolates
from broiler chicken meat, pork,
community-dwelling humans
and UTI patients. Lanes with
meat isolates are underlined.
Lanes 12 broiler chicken meat
isolates (76330048 and
763301910, respectively), lane
3 UTI isolate (357-a), lane 4
pork isolate (76331216), and
lane 5 community-dwelling
human isolate (2030c04). The
relatedness of all strains within
the individual gene profiles are
indicated in Table 2
Eur J Clin Microbiol Infect Dis
the six community-dwelling humans. Further, we do not have
information on any epidemiological link between the meat
isolates and UTI patients although consumption is likely.
Finally, the UTI isolates were from one Danish region only.
Strengths include the geographically and temporally matched
isolates from animals, meat, and humans, the knowledge of
the extensive genotypic background of the isolates, the
epidemiological information on community-dwelling human
isolates, and the in vivo model of UTI.
In conclusion, we have demonstrated closely clonally
related meat, community-dwelling human and UTI isolates
by the highly discriminating typing method, PFGE. In
addition, we have clearly demonstrated that a broiler
chicken isolate, meat and community-dwelling human
isolates are virulent in the mouse model of ascending
UTI. This is, to our knowledge, the first study to show
possible clonally related isolates from UTI patients,
community-dwelling humans, animals and meat by viru-
lence gene profile, PFGE types and in vivo virulence
characteristics, thus providing solid evidence that UTI is at
times a zoonosis. The possible and closely related
community-dwelling human and UTI isolates may indicate
Fig. 2 Bacterial counts in urine,
bladder, and kidneys of mice
killed 72 hours after inoculation
with the different B2 E. coli
strains from healthy production
animals or meat. Each point
represents the colony counts
from one mouse. The small
horizontal line represents the
median bacterial count. The
dotted line indicates the
detection limit
Eur J Clin Microbiol Infect Dis
a point source spread, e.g. through contaminated meat.
Further studies determining the proportion of animal attribut-
ed UTIs are essential to understand the actual impact of these
isolates. Depending on the impact, different measures might
be necessary, e.g. application of hazard analysis and critical
control point (HACCP) in the animal production and
slaughter, guidelines for handling and separation of specific
food items to avoid cross-contamination of ready-to-eat food,
and consumers implementing stricter hygiene routines in
kitchens or elsewhere.
Acknowledgments Frank Hansen, Karin S. Pedersen, Frederikke R.
Petersen, Leila Borggild, Jytte M. Andersen, and Dorte Truelsen are
thanked for excellent technical assistance. This study was supported
by The Danish Research Council (grant no. 2101-05-001). This work
is part of the Danish Integrated Antimicrobial Resistance Monitoring
and Research Programme (DANMAP) and the Marie Curie program
Training Risk-Assessment In Non-human Antibiotic Usage
(TRAINAU).
Declaration of interest All authors declare that they have no conflicts
of interest.
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