International Journal of Medical Microbiology 302 (2012) 129–134

Contents lists available at SciVerse ScienceDirect

International Journal of Medical Microbiology

journal homepage: www.elsevier.de/ijmm

Virulence factors and phylogenetic grouping of Escherichia coli isolates from

patients with bacteraemia of urinary tract origin relate to sex and hospital- vs.
community-acquired origin夽
Line Skjøt-Rasmussen a,∗ , Karen Ejrnæs b , Bettina Lundgren c , Anette M. Hammerum a ,
Niels Frimodt-Møller d
a
Department of Microbiological Surveillance and Research, Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen S, Denmark
b
Department of Pathology, Herlev Hospital, 75 Herlev Ringvej, 2730 Herlev, Denmark
c
The Centre of Diagnostic Investigations, Rigshospitalet, 9 Blegdamsvej, 2100 Copenhagen, Denmark
d
Department of Clinical Microbiology, Hvidovre Hospital, 30 Kettegård Allé, 2650 Hvidovre, Denmark

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

Article history: Worldwide, Escherichia coli is a leading cause of bloodstream infections. Bacteraemia cases in both
Received 27 April 2011 community- and hospital-acquired infections are often due to E. coli, and it is a major cause of mortality
Received in revised form 6 March 2012 from these infections. These invasive infections are primarily due to extraintestinal pathogenic E. coli
Accepted 10 March 2012
(ExPEC), possessing a variety of virulence factors (VFs). The pathogenesis of E. coli bloodstream infections
is largely unknown, which is why preventive measures are lacking. We studied 196 epidemiologically
Keywords:
and clinically well-characterized E. coli isolates from patients with bacteraemia of urinary tract origin
Escherichia coli
according to virulence-associated genes (VAGs), phylogroups, and antimicrobial resistance, and the rela-
Extraintestinal virulence
Bacteraemia
tion of these factors to hospital- vs. community-acquired origin, sex, and mortality. We found papAH to
Bacterial pathogenesis be associated with community-acquired (CA) rather than hospital-acquired (HA) episodes, and kpsM II
UTI and hlyD to be associated with HA rather than CA episodes. papAH and iss were associated with females,
and iroN with males. Phylogroup D was associated with females. None of the variables was found to be
related to mortality. This study provides new insights into the relationships between the epidemiology
and pathogenesis of E. coli bacteraemia of urinary tract origin.
© 2012 Elsevier GmbH. All rights reserved.

Introduction The invasive E. coli infections are primarily due to extraintestinal

Escherichia coli is a leading cause of bloodstream infections
worldwide. Around 17–37% of both community- and hospital-
acquired clinically significant bloodstream infections are due to E.
coli (Russo and Johnson, 2003), and it is a major cause of mortality
from these infections (Laupland et al., 2008; Olesen et al., 1995a;
Ron, 2010; Russo and Johnson, 2003). The (economic) burden of
sepsis is major (Russo and Johnson, 2003). Despite the frequency
and importance of E. coli bloodstream infections, the pathogenesis
is largely unknown.
夽 Part of this study was presented at the 21st European Congress of Clinical Micro-
biology and Infectious Diseases, Milan, Italy, 2011, Poster 1746; and at the Society
for General Microbiology Autumn Conference, York, UK, 2011, Poster 146.
∗ Corresponding author at: Department of Microbiological Surveillance and
Research, Statens Serum Institut, Build. 47/217, 5 Artillerivej, 2300 Copenhagen S,
Denmark. Tel.: +45 3268 3191; fax: +45 3268 3231.
E-mail address: lbs@ssi.dk (L. Skjøt-Rasmussen).
pathogenic E. coli (ExPEC) which often originate from the urinary
tract (uropathogenic E. coli, UPEC) (Olesen et al., 1995b). ExPEC pos-
sess a variety of virulence factors (VFs) responsible for pathogenesis
outside the gastrointestinal tract, i.e. they are classically described
as necessary to overcome host defences, invade host tissues, and to
trigger a local inflammatory response. The VFs belong to various
functional groups e.g. adhesins (e.g. P fimbriae, S and F1C fim-
briae), toxins (e.g. hemolysin and cytotoxic necrotizing factor), and
iron acquisition systems (e.g. the aerobactin system) (Johnson and
Russo, 2002; Russo and Johnson, 2000). The formation of biofilm
can be considered another pathogenic mechanism, allowing strains
to persist in e.g. the genitourinary or intestinal tract and hindering
the eradication of organisms (Donlan and Costerton, 2002). Human
ExPEC strains derive principally from phylogenetic groups B2 and
D (Johnson and Russo, 2002; Johnson and Stell, 2000; Russo and
Johnson, 2000).
Bacterial VFs contribute to the occurrence of bacteraemia in
humans (Jaureguy et al., 2007; Tseng et al., 2002; Wang et al., 2002),
and they may also influence the outcome of this infection (Hekker
et al., 2000). Besides VFs, host factors are also major determinants

1438-4221/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ijmm.2012.03.002
130 L. Skjøt-Rasmussen et al. / International Journal of Medical Microbiology 302 (2012) 129–134

of outcome of infection (Jaureguy et al., 2007; Lefort et al., 2011). Table 1

Virulence-associated genes and phylogenetic groups of E. coli bacteraemia isolates,
Non-presence of VFs such as adhesins has been shown to relate
grouped according to hospital- vs. community-acquired origin.
to infections in patients with immune suppression (Maslow et al.,
1993). However, few studies have investigated the decisive VFs Category, trait Occurrence of trait, no. (%) of isolates
involved when uropathogenic E. coli strains invade the bloodstream Total Hospital- Community-
(Johnson and Stell, 2000; Moreno et al., 2005; Rijavec et al., 2008). acquired acquired
Due to the high antimicrobial resistance of ExPEC strains, preven- (HA) (CA)
(n = 196) (n = 21) (n = 175)
tive measures are needed. In order to identify and develop new
targets for antimicrobial agents or develop a vaccine, it is neces- Virulence-associated genes
sary to understand the pathogenesis and virulence of bacteraemic Adhesins
afa/draBC 5 (3) 2 (10) 3 (2)
E. coli. The aim of the study was to generate knowledge about the bmaE 6 (3) 0 (0) 6 (3)
characteristics of E. coli causing urinary tract bacteraemia. We thus fimH 193 (98) 21 (100) 172 (98)
studied 196 epidemiologically and clinically well-characterized focG 44 (22) 8 (38) 36 (21)
E. coli isolates from patients with bacteraemia of urinary tract ori- gafD 2 (1) 0 (0) 2 (1)
iha 98 (50) 9 (43) 89 (51)
gin according to virulence-associated genes (VAGs), phylogroups,
papAH 140 (71) 10 (48) 130 (74)
and antimicrobial resistance, and the relation of these factors to sfa/focDE 66 (34) 13 (62) 53 (30)
hospital- vs. community-acquired origin, sex, and mortality. Biofilm-related
agn43 177 (90) 18 (86) 159 (91)
Materials and methods agn43a (CFT073) 68 (35) 10 (48) 58 (33)
agn43b (CFT073) 57 (29) 10 (48) 47 (27)
agn43 (K12) 59 (30) 9 (43) 50 (29)
Bacterial strains and patients Iron uptake
chuA 175 (89) 18 (86) 157 (90)
A collection of 196 E. coli isolates from the blood of 195 adult fyuA 180 (92) 19 (90) 161 (92)
ireA 65 (33) 4 (19) 61 (35)
patients with both bacteraemia and bacteriuria was studied. From
iroN 110 (56) 15 (71) 95 (54)
one patient, 2 distinct E. coli blood isolates were cultured. Iso- iutA 147 (75) 14 (67) 133 (76)
lates were collected from January 2003 through May 2005 from Protectins
all patients older than 18 years admitted to Amager Hospital, Bis- iss 46 (23) 4 (19) 42 (24)
pebjerg Hospital, Frederiksberg Hospital, or Hvidovre Hospital in kpsM II 163 (83) 18 (86) 145 (83)
kpsM II K2 169 (86) 18 (86) 151 (86)
Copenhagen. Isolates represent episodes of E. coli bacteraemia with kpsMT III 1 (<1) 0 (0) 1 (<1)
bacteriuria, where a positive E. coli urine culture was performed traT 134 (68) 11 (52) 123 (70)
±3 days within the blood culture date. Episodes were considered Toxins
community-acquired (CA) when they were diagnosed within the cdtB 18 (9) 1 (5) 17 (10)
cnf1 56 (29) 10 (48) 46 (26)
first 48 h of hospitalization and were considered hospital-acquired
hlyD 66 (34) 12 (57) 54 (31)
(HA) after this period. E. coli identification was performed according sat 88 (45) 7 (33) 81 (46)
to standard procedures. Isolates were stored at −80 ◦ C until use. The Miscellaneous
study protocol was approved by the Scientific Ethics Committee for ibeA 23 (12) 2 (10) 21 (12)
the Capital Region of Denmark [(KF) 01 2006-6371)]. malX 128 (65) 14 (67) 114 (65)
usp 133 (68) 16 (76) 117 (67)
Phylogenetic groups
Epidemiological and clinical data A 8 (4) 0 (0) 8 (5)
B1 8 (4) 2 (10) 6 (3)
Epidemiological (age, sex), clinical (date of hospital admission, B2 131 (67) 15 (71) 116 (66)
D 44 (22) 3 (14) 41 (23)
date of death), and laboratory (blood and urine culture dates) data
Non-typeable 5 (3) 1 (5) 4 (2)
for each episode were extracted from the patients’ medical records
and the hospital administrative database.

brain endothelium), malX (pathogenicity-associated island marker

Virulence genotyping
of CFT073), and usp (uropathogenic specific protein) (see also
Table 1). chuA was detected by the triplex PCR for phylogenetic
The isolates were screened for the presence of 29 VAGs with
groups (Clermont et al., 2000). The rest of the genes were detected
known or suspected relevance to ExPEC pathogenesis, as described
by using a modification of the multiplex PCR protocol described by
by Ejrnæs et al. (2011). The VAGs represent 6 functional groups: (1)
Johnson and Stell, 2000 (Ejrnæs et al., 2011). There were 6 primer
8 adhesins: afa/draBC (Dr. binding adhesin), bmaE (blood group M
pools; the pools, and primers used for detection of the VAGs are pre-
fimbriae), fimH (type 1 fimbriae), focG (F1C fimbriae), gafD (G fim-
sented in Suppl. Table 1. Nine strains were used as positive controls
briae), iha (iron-regulated gene A homologue adhesin, siderophore
in the PCR screenings: E. coli 2H16 (primer pools 1, 3–5) (Johnson
receptor), papAH (P fimbriae), and sfa/focDE (S fimbriae, F1C fim-
et al., 2000); E. coli 2H25 (primer pool 2) (Johnson et al., 2000); E. coli
briae); (2) 4 biofilm-related VAGs: agn43 (Antigen 43, common),
PM9, RS218, J96, L31, and V27 (primer pools 1–5) (Johnson et al.,
agn43aCFT073 (Antigen 43, allele a CFT073), agn43bCFT073 (Anti-
1997, 2000, 2001a,b); and E. coli MG1655 and CFT073 (primer pools
gen 43, allele b CFT073), and agn43K12 (Antigen 43, allele K12);
4–6) (Restieri et al., 2007). E. coli P679-54 (aka JJ055) and PCR water
(3) 5 iron uptake VAGs: chuA (heme receptor), fyuA (yersiniabactin
(Invitrogen A/S, Taastrup, Denmark) were used as negative controls
siderophore receptor), ireA (iron-regulated element, siderophore
in the reactions. PCR conditions were as described in Ejrnæs et al.
receptor), iroN (salmochelin siderophore receptor), and iutA
(2011).
(aerobactin siderophore receptor); (4) 5 protectins: iss (increased
serum survival), kpsM II (group II capsule), kpsM II K2 (group II cap-
sule, K2), kpsM III (group III capsule), and traT (serum resistance); Phylogenetic analysis
(5) 4 toxins: cdtB (cytolethal distending toxin), cnf1 (cytotoxic
necrotizing factor 1), hlyD (alpha hemolysin), and sat (secreted The phylogenetic background (A, B1, B2, and D) of all isolates
autotransporter toxin); (6) 3 miscellaneous VAGs: ibeA (invasion of was determined by triplex PCR using 3 DNA markers (Clermont
 L. Skjøt-Rasmussen et al. / International Journal of Medical Microbiology 302 (2012) 129–134
et al., 2000). Results obtained allowed classification of the isolates
into the 4 major E. coli phylogenetic lineages or non-typeable (NT)
isolates according to Gordon et al. (2008).
Antimicrobial resistance
Minimum inhibitory concentrations (MICs) for ampicillin, cef-
podoxime, ceftiofur, chloramphenicol, ciprofloxacin, gentamicin,
nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim
were determined for the isolates by a microbroth dilution
method as defined by the Clinical and Laboratory Standards Insti-
tute (CLSI). E. coli ATCC 25922 was used for quality control.
Results were interpreted according to the European Commit-
tee on Antimicrobial Susceptibility Testing (EUCAST) guidelines
(http://www.eucast.org), except for sulfamethoxazole and tetra-
cycline which were interpreted according to CLSI (CLSI, 2008).
For ciprofloxacin a breakpoint of ≥0.125 mg/l was used (Aarestrup
et al., 2003). ESBL and/or AmpC phenotypes were tested with a
combination disk method (Rosco NeoSensitabs, Rosco, Taastrup,
Denmark).
Statistical analysis
Comparisons of proportions were based on the chi-square
test or, when expected numbers were less than 5, Fisher’s exact
test (two-tailed). A P-value <0.05 was considered statistically
significant. Forward and backward multiple regression analysis
was performed with mortality, sex, or hospital- vs. community-
acquired origin as the dependent variable and bacterial traits
(VAGs and antimicrobial resistance) as the predictor variables using
Statistica version 7. A similar analysis was performed with the indi-
vidual phylogenetic groups as the dependent variable and VAGs as
the predictor variables. Excluded from these analyses were those
VAGs with a very low or high prevalence (afa/draBC, bmaE, fimH,
gafD, and kpsM III) and chuA because of the relation to the phylo-
genetic groups; the NT phylogenetic group was also excluded from
the analysis due to its low prevalence. Furthermore, clustering of
the E. coli according to the presence and absence of the same fac-
tors was performed using Statistica version 7 in order to study the
45
40
35
% resistant isolates
30
25
20
15
10
Hospital-acquired (n = 21)
Fig. 1. Antimicrobial resistance (%) in E. coli bacteraemia isolates, grouped according to hospital- vs. community-acquired origin.
131
possible association of VAGs with mortality, sex, or hospital- vs.
community-acquired origin.
Results
Individual data are presented in Suppl. Table 2.
Clinical characteristics
The median age of the 195 patients was 79 years (range 19–102
years); 133 patients (68%) were female, 62 (32%) were male.
Twenty-one infection episodes (11%) were considered HA and 174
episodes (89%) CA. Total 30-day mortality was 12%, 24% among
HA episodes and 10% among CA episodes. Fewer males (5%) than
females (15%) died within 30 days from culture date.
Distribution of VAGs
The occurrence of VAGs among the E. coli isolates ranged from
<1% (kpsMT III) to 98% (fimH) (Table 1). One to twenty-two VAGs
were detected per E. coli isolate.
Phylogenetic distribution
Isolates belonging to all 4 major phylogenetic lineages and NTs
were found. Most isolates belonged to phylogenetic lineages B2
(n = 131, 67%) and D (n = 44, 22%), and a few A, B1, and NT isolates
were found (Table 1).
Antimicrobial resistance
Eighty-nine isolates (45%) were fully antimicrobial susceptible.
The highest resistance rates were detected among CA isolates, while
the HA isolates were the most susceptible (Fig. 1). However, there
were no statistically significant differences in antimicrobial resis-
tance rates neither among CA vs. HA isolates nor among isolates
from patients dying vs. not dying within 30 days nor among isolates
from females vs. males. Three isolates were cefpodoxime-resistant;
none of these produced ESBL.
Community-acquired (n = 175)
132 L. Skjøt-Rasmussen et al. / International Journal of Medical Microbiology 302 (2012) 129–134
Association with hospital- vs. community-acquired origin
Multiple regression analysis found papAH to be independently
and significantly associated with CA episodes (74%) rather than HA
episodes (48%) (P = 0.018). On the other hand, kpsM II (P = 0.034) and
hlyD (P = 0.044) were found to be associated with HA (86% and 57%,
respectively) rather than CA (83% and 31%, respectively) episodes
(Table 1). No phylogenetic groups were found to be associated
with hospital- vs. community-acquired origin. E. coli associated
with hospital- vs. community-acquired origin did not segregate in
separate clusters in relation to VAGs.
Association with sex
By multiple regression, 3 VAGs were found to be independently
and significantly associated with either female or male patients. iss
(P = 0.014) and papAH (P = 0.023) were found to be associated with
females (28% and 75%, respectively) rather than males (13% and
65%, respectively), whereas iroN was found to be associated with
males (65%) rather than females (52%) (P = 0.026). Phylogroup D was
found to be associated with females (28%) rather than males (10%)
(P = 0.004, chi-squared test). E. coli associated with either female vs.
male patients did not segregate in separate clusters in relation to
VAGs.
Association with mortality
Multiple regression analysis showed that no VAGs or antimicro-
bial resistance were associated with mortality. Also, mortality was
not associated with any phylogenetic groups. E. coli from patients
dying within 30 days of culture did not segregate in separate clus-
ters in relation to VAGs. No clear distinction based on the presence
and absence of VAGs could thus be made between isolates from
patients dying within 30 days as compared to isolates from patients
not dying within 30 days.
Association between phylogenetic groups and VAGs
By multiple regression analysis, fyuA (P = 0.002) and
agn43aCFT073 (P = 0.011) were found to be positively associ-
ated with phylogroup A, whereas kpsM II K2 (P = 0.003) and iha
(P = 0.007) were negatively associated with phylogroup A. fyuA
(P = 0.010) and iha (P = 0.048) were negatively associated with
phylogroup B1. usp (P < 0.001) and agn43 (P = 0.035) were posi-
tively associated with phylogroup B2, whereas sfa/focDE (P = 0.013)
was negatively associated with phylogroup B2. iha (P < 0.001),
sfa/focDE (P = 0.004), iss (P = 0.007), ibeA (P = 0.016), and kpsM II K2
(P = 0.046) were positively associated with phylogroup D, whereas
usp (P < 0.001), agn43aCFT073 (P < 0.001), iroN (P = 0.004), and hlyD
(P = 0.039) were negatively associated with phylogroup D.
Discussion
We present a thorough study of 196 epidemiologically and clini-
cally well-characterized E. coli bloodstream isolates of urinary tract
origin. The isolates were analyzed for a broad range of virulence
genotypes, their phylogenetic background, resistance to various
antimicrobial agents, and the relationship of these factors to patient
mortality, hospital- vs. community-acquired origin, and patient sex
was explored. To our knowledge, our study is the first to study
the presence of Ag43 and its allelic variants of UPEC strain CFT073
(agn43aCFT073 and agn43bCFT073) in clinical E. coli bacteraemia
isolates.
In studies of E. coli bacteraemia, a wide variability in case-fatality
rates has been observed, ranging broadly from 5% to 30% (Laupland
et al., 2008). As compared to a study by Olesen et al. (1995b),
in which a 4-week mortality of 21% among E. coli bacteraemia
episodes in a Danish university hospital was found, our observed
overall 30-day mortality of 12% among E. coli bacteraemia cases
was low.
Only few previous studies have investigated associations
between host and bacterial determinants and the outcome of E. coli
bacteraemia in humans (Jaureguy et al., 2007; Lefort et al., 2011). To
the best of our knowledge, no such studies have investigated asso-
ciations specifically among E. coli bacteraemia cases with a urinary
tract origin.
High virulence potential is typically found in strains from
immunocompetent patients, the virulence characteristics helping
the bacteria overcome host defences (Jaureguy et al., 2007). In
accordance with other studies, papAH coding for P fimbriae was
found to be associated with CA rather than HA episodes (Jaureguy
et al., 2007). HA bacteraemias are typically caused by isolates with
a lower virulence potential since the VF products are less neces-
sary when host defences are impaired (Cooke et al., 2010). In our
study, however, the 2 VAGs kpsM II and hlyD, coding for group II cap-
sule and alpha hemolysin, respectively, were found to be associated
with HA rather than with CA episodes. Thus, our results indicate
that both CA and HA infections can be due to virulent strains and
that these hospitalized patients are perhaps not as immunocom-
promised as assumed. Yet, discrepant findings regarding the effect
of host compromise on the prevalence of various bacterial traits
have been described (Johnson et al., 2002).
Urinary tract infection (UTI) is most common among women,
but is also a problem among men. The bacterial characteristics
required for establishing infection are supposedly different among
the sexes. papAH and iss, coding for P fimbriae and increased
serum survival, respectively, were significantly associated with
female patients, whereas iroN, coding for a siderophore recep-
tor, was associated with male patients. iroN has previously been
shown to be associated with urinary-source E. coli in men (Johnson
et al., 2005a). Thus, our results support the hypothesis that the UTI
pathogenesis differs among women and men, being reflected in
part by differing traits required to establish an infection in the 2
sexes.
No VAGs were found to be associated with mortality. The pro-
portion of isolates from patients dying within 30 days of culture
was low which could explain the lack of VAGs predicting mortal-
ity. Jaureguy et al. (2007) found papGIII to be associated with an
increased fatal outcome of E. coli sepsis. In the present study, we
did not screen for papGIII as being associated with E. coli causing
prostatitis (Ruiz et al., 2002). Thus, our study did not succeed to
associate particular bacterial determinants with the outcome of
E. coli causing urinary tract bacteraemia; however, the results still
provide new insights into the association of VAGs with the outcome
of bacteraemia of urinary tract origin. As shown by others (Jaureguy
et al., 2007; Lefort et al., 2011), this result could also indicate that
host or iatrogenic factors may be more important than bacterial
factors for the course of E. coli bacteraemia.
Most isolates belonged to phylogenetic lineages B2 and D. This
agrees with most other studies concerning phylogenetic groups in
uropathogenic (Johnson et al., 2005b; Moreno et al., 2008) and bac-
teraemic E. coli (Johnson et al., 2002; Moreno et al., 2005; Sannes
et al., 2004). Phylogroup D isolates were associated with females
rather than males. We have no explanation for this observation,
but are investigating a possible spread of clones associated with
phylogroup D.
VAGs were found to be positively and/or negatively associated
with all phylogroups. Positive VAG predictors were identified espe-
cially for phylogroups B2 and D, confirming that phylogroup B2 and
to a lesser extent phylogroup D are the groups which include potent
human ExPEC isolates causing among others UTI and bacteraemia
(Johnson and Russo, 2002; Moreno et al., 2005, 2008).
 L. Skjøt-Rasmussen et al. / International Journal of Medical Microbiology 302 (2012) 129–134
The E. coli isolates were collected in Denmark, a country char-
acterized by a low level of antimicrobial resistance, and the level
of antimicrobial resistance among our isolates was also found to be
low. In our study, none of the isolates were ESBL-producing. This
is in line with the fact that ESBL-producing E. coli had a very low
prevalence during the sample collection period. The higher level of
antimicrobial resistance among CA isolates than among HA isolates
indicates that resistance in E. coli is to a higher degree correlated to
the use of antimicrobials outside than in hospitals, i.e. in primary
health care and for animal therapy. This follows in line with the
fact that more than 90% of antibiotic consumption in humans takes
place in primary health care (DANMAP, 2010).
The results of this study are strengthened by the size of the strain
collection as well as the quality of the clinical data. However, inter-
dependence or close linkage of bacterial traits, e.g. on pathogenicity
islands, and not the trait itself can be the reason for the finding
of virulence determinants with a significant correlation to clinical
variables.
More studies on selected strains are needed in order to examine
the virulence potential of such strains in further detail. Also, further
sequencing of E. coli strains causing bacteraemia could aid in the
finding and description of genes or gene clusters which are pecu-
liar for E. coli causing bacteraemia and which were not included
in our elaborate but perhaps selective study of possible VFs. Also,
the lack of association of specific VFs for mortality in E. coli bacter-
aemia should lead to more intensive search for relevant host factors,
which would further aid in understanding this serious disease and
its successful treatment.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
Karin Sixhøj Pedersen, Frank Hansen, and Alexandra Medina
at Statens Serum Institut and staff at the Department of Clini-
cal Microbiology at Hvidovre Hospital are thanked for excellent
technical assistance. This study was supported by the Danish Med-
ical Research Council (grant number 22-02-0373 ct/mp) and is
part of the Danish Integrated Antimicrobial Resistance Monitoring
and Research Programme (DANMAP). We wish to thank Flemming
Scheutz for providing control strains and James R. Johnson for pro-
viding technical advices and control strains.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.ijmm.2012.03.002.
References
Aarestrup, F.M., Wiuff, C., Mølbak, K., Threlfall, J.E., 2003. Is it time to change fluoro-
quinolone breakpoints for Salmonella spp.? Antimicrob. Agents Chemother. 47,
827–829.
Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the
Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558.
CLSI, 2008. Performance Standards for Antimicrobial Susceptibility Testing, 18th
Informational Supplement, M100-S18. Clinical and Laboratory Standards Insti-
tute, Wayne, PA.
Cooke, N.M., Smith, S.G., Kelleher, M., Rogers, T.R., 2010. Major differences exist
in frequencies of virulence factors and multidrug resistance between commu-
nity and nosocomial Escherichia coli bloodstream isolates. J. Clin. Microbiol. 48,
1099–1104.
DANMAP, 2010. Use of antimicrobial agents and occurrence of antimicrobial
resistance in bacteria from food animals, food and humans in Denmark.
ISSN 1600-2032. http://www.danmap.org/pdfFiles/Danmap 2010.pdf (accessed
08.12.11).
133
Donlan, R.M., Costerton, J.W., 2002. Biofilms: survival mechanisms of clinically rel-
evant microorganisms. Clin. Microbiol. Rev. 15, 167–193.
Ejrnæs, K., Stegger, M., Reisner, A., Ferry, S., Monsen, T., Holm, S.E., Lundgren, B.,
Frimodt-Møller, N., 2011. Characteristics of Escherichia coli causing persistence
or relapse of urinary tract infections: phylogenetic groups, virulence factors and
biofilm formation. Virulence 2, 528–537.
Gordon, D.M., Clermont, O., Tolley, H., Denamur, E., 2008. Assigning Escherichia coli
strains to phylogenetic groups: multi-locus sequence typing versus the PCR
triplex method. Environ. Microbiol. 10, 2484–2496.
Hekker, T.A.M., Groeneveld, A.B.J., Simoons-Smit, A.M., de Man, P., Connell, H.,
MacLaren, D.M., 2000. Role of bacterial virulence factors and host factors in the
outcome of Escherichia coli bacteraemia. Eur. J. Clin. Microbiol. Infect. Dis. 19,
312–316.
Jaureguy, F., Carbonnelle, E., Bonacorsi, S., Clec’h, C., Casassus, P., Bingen, E., Picard,
B., Nassif, X., Lortholary, O., 2007. Host and bacterial determinants of ini-
tial severity and outcome of Escherichia coli sepsis. Clin. Microbiol. Infect. 13,
854–862.
Johnson, J.R., Delavari, P., O’Bryan, T.T., 2001a. Escherichia coli O18:K1:H7 isolates
from patients with acute cystitis and neonatal meningitis exhibit common phy-
logenetic origins and virulence factor profiles. J. Infect. Dis. 183, 425–434.
Johnson, J.R., Kuskowski, M.A., Gajewski, A., Soto, S., Horcajada, J.P., Jimenez de
Anta, M.T., Vila, J., 2005b. Extended virulence genotypes and phylogenetic back-
ground of Escherichia coli isolates from patients with cystitis, pyelonephritis, or
prostatitis. J. Infect. Dis. 191, 46–50.
Johnson, J.R., Kuskowski, M.A., O’Bryan, T.T., Maslow, J.N., 2002. Epidemiolog-
ical correlates of virulence genotype and phylogenetic background among
Escherichia coli blood isolates from adults with diverse-source bacteremia. J.
Infect. Dis. 185, 1439–1447.
Johnson, J.R., Russo, T.A., 2002. Extraintestinal pathogenic Escherichia coli: “The other
bad E. coli”. J. Lab. Clin. Med. 139, 155–162.
Johnson, J.R., Russo, T.A., Scheutz, F., Brown, J.J., Zhang, L., Palin, K., Rode, C., Bloch,
C., Marrs, C.F., Foxman, B., 1997. Discovery of disseminated J96-like strains of
uropathogenic Escherichia coli O4:H5 containing genes for both PapG(J96) (class
I) and PrsG(J96) (class III) Gal(alpha1–4)Gal-binding adhesins. J. Infect. Dis. 175,
983–988.
Johnson, J.R., Russo, T.A., Tarr, P.I., Carlino, U., Bilge, S.S., Vary, J.C., Stell, A.L., 2000.
Molecular epidemiological and phylogenetic associations of two novel puta-
tive virulence genes, iha and iroN (E. coli), among Escherichia coli isolates from
patients with urosepsis. Infect. Immun. 68, 3040–3047.
Johnson, J.R., Scheutz, F., Ulleryd, P., Kuskowski, M.A., O’Bryan, T.T., Sandberg, T.,
2005a. Phylogenetic and pathotypic comparison of concurrent urine and rectal
Escherichia coli isolates from men with febrile urinary tract infection. J. Clin.
Microbiol. 43, 3895–3900.
Johnson, J.R., Stell, A.L., 2000. Extended virulence genotypes of Escherichia coli strains
from patients with urosepsis in relation to phylogeny and host compromise. J.
Infect. Dis. 181, 261–272.
Johnson, J.R., Stell, A.L., Kaster, N., Fasching, C., O’Bryan, T.T., 2001b. Novel molecular
variants of allele I of the Escherichia coli P fimbrial adhesin gene papG. Infect.
Immun. 69, 2318–2327.
Laupland, K.B., Gregson, D.B., Church, D.L., Ross, T., Pitout, J.D., 2008. Incidence,
risk factors and outcomes of Escherichia coli bloodstream infections in a large
Canadian region. Clin. Microbiol. Infect. 14, 1041–1047.
Lefort, A., Panhard, X., Clermont, O., Woerther, P.-L., Branger, C., Mentré, F., Fantin,
B., Wolff, M., Denamur, E., 2011. Host factors and portal of entry outweigh bac-
terial determinants to predict the severity of Escherichia coli bacteremia. J. Clin.
Microbiol. 49, 777–783.
Maslow, J.N., Mulligan, M.E., Adams, K.S., Justis, J.C., Arbeit, R.D., 1993. Bacterial
adhesins and host factors: role in the development and outcome of Escherichia
coli bacteremia. Clin. Infect. Dis. 17, 89–97.
Moreno, E., Andreu, A., Pigrau, C., Kuskowski, M.A., Johnson, J.R., Prats, G.,
2008. Relationship between Escherichia coli strains causing acute cystitis in
women and the fecal E. coli population of the host. J. Clin. Microbiol. 46,
2529–2534.
Moreno, E., Planells, I., Prats, G., Planes, A.M., Moreno, G., Andreu, A., 2005. Compar-
ative study of Escherichia coli virulence determinants in strains causing urinary
tract bacteremia versus strains causing pyelonephritis and other sources of bac-
teremia. Diagn. Microbiol. Infect. Dis. 53, 93–99.
Olesen, B., Kolmos, H.J., Orskov, F., Orskov, I., 1995a. A comparative study of noso-
comial and community-acquired strains of Escherichia coli causing bacteraemia
in a Danish University Hospital. J. Hosp. Infect. 31, 295–304.
Olesen, B., Kolmos, H.J., Orskov, F., Orskov, I., Gottschau, A., 1995b. Bacteraemia due
to Escherichia coli in a Danish university hospital, 1986–1990. Scand. J. Infect.
Dis. 27, 253–257.
Restieri, C., Garriss, G., Locas, M.C., Dozois, C.M., 2007. Autotransporter-encoding
sequences are phylogenetically distributed among Escherichia coli clinical iso-
lates and reference strains. Appl. Environ. Microbiol. 73, 1553–1562.
Rijavec, M., Müller-Premru, M., Zakotnik, B., Zgur-Bertok, D., 2008. Virulence factors
and biofilm production among Escherichia coli strains causing bacteraemia of
urinary tract origin. J. Med. Microbiol. 57, 1329–1334.
Ron, E.Z., 2010. Distribution and evolution of virulence factors in septicemic
Escherichia coli. Int. J. Med. Microbiol. 300, 367–370.
Ruiz, J., Simon, K., Horcajada, J.P., Velasco, M., Barranco, M., Roig, G., Moreno-
Martinez, A., Martinez, J.A., Jiménez de Anta, T., Mensa, J., Vila, J., 2002.
Differences in virulence factors among clinical isolates of Escherichia coli causing
cystitis and pyelonephritis in women and prostatitis in men. J. Clin. Microbiol.
40, 4445–4449.
134 L. Skjøt-Rasmussen et al. / International Journal of Medical Microbiology 302 (2012) 129–134
Russo, T.A., Johnson, J.R., 2000. Proposal for a new inclusive designation for
extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J. Infect. Dis. 181,
1753–1754.
Russo, T.A., Johnson, J.R., 2003. Medical and economic impact of extraintestinal
infections due to Escherichia coli: focus on an increasingly important endemic
problem. Microbes Infect. 5, 449–456.
Sannes, M.R., Kuskowski, M.A., Owens, K., Gajewski, A., Johnson, J.R., 2004. Viru-
lence factor profiles and phylogenetic background of Escherichia coli isolates
from veterans with bacteremia and uninfected control subjects. J. Infect. Dis.
190, 2121–2128.
Tseng, C.-C., Wu, J.-J., Liu, H.-L., Sung, J.-M., Huang, J.-J., 2002. Roles of host and
bacterial virulence factors in the development of upper urinary tract infection
caused by Escherichia coli. Am. J. Kidney Dis. 39, 744–752.
Wang, M.-C., Tseng, C.-C., Chen, C.-Y., Wu, J.-J., Huang, J.-J., 2002. The role of bac-
terial virulence and host factors in patients with Escherichia coli bacteremia
who have acute cholangitis or upper urinary tract infection. Clin. Infect. Dis.
35, 1161–1166.