BRIEF REPORT
Escherichia coli Isolates Causing
Bacteremia via Gut Translocation
and Urinary Tract Infection in Young
Infants Exhibit Different Virulence
Genotypes
Farah Mahjoub-Messai,1,3 Philippe Bidet,1,3 Valérie Caro,4 Laure Diancourt,4
Valérie Biran,2 Yannick Aujard,2,3 Edouard Bingen,1,3 and Stéphane
Bonacorsi1,3
1Service
de Microbiologie and Laboratoire Associé au Centre National de
Référence de Escherichia coli, 2Service de Néonatologie, Hôpital Robert Debré,
Assistance Publique–Hôpitaux de Paris, 3Equipe d'accueil EA 3105, Université Paris
Diderot, and 4Institut Pasteur, Genotyping of Pathogens and Public Health,
Paris, France
gut translocation (GT). In LOI, E. coli may be acquired from the
mother or the environment and may enter the bloodstream
from the urinary tract or by GT.
Several mechanisms that promote the translocation of
indigenous bacteria from the gut have been identified, such as
intestinal bacterial overgrowth, deficiencies in host immune
defenses, and intestinal mucosal barrier damage [2]. E. coli
strains that cause bacteremia by GT have rarely been investigated
[3] and have not specifically been compared with urinary tract
infection (UTI) strains. We therefore compared the genetic
background and virulence determinants of 100 E. coli isolates
from young infants with bacteremia due either to UTI or to GT,
to identify genetic traits that may contribute to the pathophysiological steps leading to E. coli bacteremia in this population.
MATERIALS AND METHODS
Escherichia coli bacteremia in young infants may arise via
either urinary tract infection or gut translocation (GT).
E. coli GT isolates have rarely been investigated. Molecular
analysis of 100 E. coli isolates recovered from bacteremic
infants revealed that GT isolates had multilocus sequence
types similar to those of urosepsis isolates but different
prevalences of PapGII adhesin, TcpC protectin, and ibeA
invasin. Compared with late-onset GT isolates, early-onset
isolates were associated with significantly different rates of
the conserved virulence plasmidic region common to human
and avian pathogenic strains and a-hemolysin. We identified
genetic determinants potentially involved in specific pathophysiological steps preceding E. coli bloodstream invasion.
Escherichia coli is the leading cause of bacteremia in infants aged
,3 months [1]. During this age period, pediatricians usually
distinguish early-onset infections (EOIs), which occur during
the first 3 days of life, from late-onset infections (LOIs). In EOI,
E. coli is acquired from the mother before or during birth. In this
situation E. coli generally colonizes the genital tract before
entering the digestive tract and spreading to the bloodstream by
Received 16 October 2010; accepted 31 January 2011.
Potential conflicts of interest: none reported.
Correspondence: Stéphane Bonacorsi, PhD, Service de Microbiologie, Hôpital Robert Debré,
48 Boulevard Sérurier, 75019 Paris ([email protected]).
The Journal of Infectious Diseases 2011;203:1844–9
Ó The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases
Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@
oup.com
0022-1899 (print)/1537-6613 (online)/2011/20312-0019$14.00
DOI: 10.1093/infdis/jir189
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We studied E. coli blood culture isolates recovered from infants
aged ,90 days who had been admitted to Robert-Debré Pediatric Hospital (Paris, France) during the period 1999–2009.
Bacteremia was considered secondary to GT when (1) an isolate
with the same virulence genotype and serogroup as the blood
isolate was cultured from gastric fluid specimens obtained from
infants with EOI or from stool specimens (.108 CFU/g of feces,
as determined on the basis of colony morphology) obtained
from those with LOI, (2) no primary site of infection was
detected, (3) urine culture results were negative (,1000 CFU/
mL) for infants aged .4 days, and (4) no congenital or acquired
digestive tract abnormalities were present. Bacteremia secondary
to UTI was defined by the presence of R100,000 colonyforming units of E. coli CFU per milliliter of clean voided or bag
specimens of urine, or R10,000 or more CFU/mL for catheterized specimens, with no other identified site of infection.
Patients with UTI underwent voiding cytourethrography, and
those found who were to have major urinary tract abnormalities
(eg, grade III to V vesicoureteral reflux, posterior urethral
valves, or pyelo-ureteric junction syndrome) were excluded
from the study.
Multilocus sequence typing (MLST) was performed in
accordance with the Achtman scheme, described at http://mlst.
ucc.ie/mlst/dbs/Ecoli. The 7 gene sequences were concatenated
for each strain (3421 letters), and a phylogenetic tree was constructed with the MEGA3.1 program from synonymous distances, using the neighbor-joining algorithm [4].
Twenty-one virulence-related factors were sought by means of
polymerase chain reaction (PCR) (Table 1). Ten virulence
Table 1. Clinical and Bacterial Characteristics of Escherichia coli Bacteremia
Portal of entry of E. coli bacteremia
Gut translocation time
Host and bacterial characteristics
Any site
(n5 100)
Urinary tract
(n5 74)
Any age
(n526)
Pa
,72 h of
life (n513)
Pb
.72 h of
life (n513)
Clinical features
Age, median days
29
40
No. (%) of male patients
77
66 (89.1)
11 (42.3)
4
,.0001
2
,.0001
5 (38.4)
Term, median weeks
39
39.5
32.6
.0002
Postconception age, median weeks
42.1
43
37.1
,.0001
Group B2
Group D
75
16
57 (77)
12 (16.2)
18 (69.2)
4 (15)
Group B1
7
4 (5.4)
Group A
2
1 (1.3)
Antigen K1
52
35 (47.2)
tcpC
23
21 (28)
,.0001
NS
24
6 (46.1)
32.6
NS
30
33
NS
33.4
NS
NS
11 (84.6)
0
NS
NS
7 (53.8)
4 (30)
3 (11.5)
NS
1 (7.6)
NS
2 (15.3)
1 (3.8)
NS
1 (7.6)
NS
0
17 (65.3)
NS
10 (77)
NS
7 (53.8)
2 (7.6)
.03
1 (7.6)
NS
1 (7.6)
9 (69)
Phylogroup, as determined by MLST
Protectins
Adhesins/invasins
papGII
72
58 (78.3)
papGIII
4
4 (5.4)
sfa/foc
23
hek/hra
.016
5 (38)
NS
0
NS
0
NS
0
18 (24)
5 (19)
NS
4 (30.7)
NS
1 (7.6)
27
219 (28)
6 (23)
NS
1 (7.6)
NS
5 (38.4)
9
2 (2.7)
7 (27)
.0002
4 (30.7)
NS
3 (23)
cnf1
15
14 (19)
1 (3.8)
NS
0
NS
1 (7.6)
hlyC
sat
29
39
23 (31)
29 (39)
6 (23)
10 (38)
NS
NS
0
3 (23)
clbN/clbB
30
24 (32.4)
6 (23)
NS
4 (30)
NS
2 (15.3)
vat
70
52 (70.2)
18 (69.2)
NS
11 (84.6)
NS
7 (53.8)
cdt
2
2 (2.7)
NS
0
iroN
65
49 (66.2)
16 (61.5)
NS
11 (84.6)
.02
5 (38.4)
fyuA
98
72 (97.2)
26 (100)
NS
13 (100)
NS
13 (100)
iucC
sitA
87
98
65 (87.8)
72 (97)
22 (84.6)
26 (100)
NS
NS
12 (92.3)
13 (100)
NS
NS
10 (77)
13 (100)
cvaA
59
46 (62.1)
13 (50)
NS
10 (76.9)
.01
3 (23)
etsC
38
26 (35.1)
12 (46.1)
NS
9 (69.2)
.02
3 (23)
iss
41
28 (37.8)
13 (50)
NS
9 (69.2)
.04
4 (30)
ompTp
41
28 (37.8)
13 (50)
NS
9 (69.2)
.04
4 (30)
hlyF
42
36
29 (39.1)
26 (35.1)
13 (50)
10 (38.4)
NS
NS
9 (69.2)
8 (61.5)
.04
.01
4 (30)
2 (15.3)
NS
8.3
ibeA
14 (56)
Toxins
0
NS
0.01
NS
0
6 (46.1)
7 (53.8)
Iron uptake system
Plasmidic determinants
CVP regionc
Virulence scored
9.64
9.7
9.46
NS
10.62
NOTE. MLST, multilocus sequence typing; NS, not significant.
a
Urinary tract infection versus gut translocation.
b
Early- vs late-onset gut translocation.
c
Conserved virulence plasmidic region, characterized by simultaneous presence of cvaA, etsC, iss, ompTp, hlyF, iucC, iroN, and sitA.
d
Mean number of virulence factors per strain.
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factor-encoding genes characteristic of extraintestinal pathogenic E. coli (ExPEC; papGII and papGIII, P fimbriae; sfa/foc,
S fimbriae; hlyC, hemolysin; cnf1, cytotoxic necrotizing factor;
iucC, aerobactin; fyuA, yersiniabactin; iroN, salmochelin; hek/
hra, haemagglutinin; and ibeA, endothelial invasin) were identified by multiplex PCR, as previously elsewhere [4]. Other toxin
genes (sat, serine protease autotransporter toxin; vat, vacuolating autotransporter toxin; cdt, cytolethal distending toxin; and
clbN/clbB colibactin) were sought by using a new multiplex PCR
method with primers described elsewhere [5–7]. PCR was performed in a 50-lL volume with 25 lL of 2X Qiagen Multiple
PCR Master Mix (Qiagen), 5 lL of 5X Q-solution, 200 nmol/L
each primer, 10 lL of distilled water, and 5 lL of bacterial lysate.
The recently described gene tcpC, which encodes TIR domaincontaining protein, was detected by PCR as described by Cirl
et al. [8]. We also investigated the distribution of genetic
determinants characteristic of virulence plasmids found in both
human ExPEC and avian pathogenic E. coli (APEC) [9, 10]. The
determinants ompTp, etsC, iss, hlyF, and cvaA were detected by
PCR as described elsewhere [10]. The presence of these genes,
together with the genes encoding salmochelin, aerobactin, and
the iron–uptake system SitABC, was considered to be a signature
of a conserved virulence plasmidic (CVP) region described in
both human ExPEC and APEC [9, 10]. Positive controls for the
virulence loci were strains CFT073, C5, and S88.
The most frequent O antigens described in ExPEC strains
isolated from young infants (O1, O2, O4, O6, O7, O16, O18,
O45, and O83) and the K1 capsular antigen were detected as
described elsewhere [4].
Proportions were compared between groups by using the
Fisher exact test, and median values were compared by use of the
Mann-Whitney U test. Student’s t test was used to compare
means. P values ,.05 were considered to denote significant
differences.
MLST identified 35 sequence types (STs). Twenty-five STs
could be affiliated with 14 ST complexes (STcs), of which STc95
was the most prevalent (38%). Three other major STcs,
accounting for R5% of the strains, were observed—namely,
STc73 (n 5 15), STc12 (n 5 7), and STc69 (n 5 6) (Figure 1).
Phylogenetic analysis of the MLST data clustered the strains into
4 major phylogenetic groups (Table 1 and Figure 1). Groups B2
and D accounted for 75% and 16% of the strains, respectively,
and nonvirulent groups (A/B1) represented ,10%. No difference between the UTI and GT strain subcollections was found in
terms of the major phylogenetic groups or STc distributions
(Table 1 and Figure 1). As described elsewhere, MLST and
serogrouping can be combined to identify clonal groups, that are
designated by a sequence O type [4]. The distribution of major
sequence O types between UTI and GT isolates was similar
(Figure 1).
Regarding virulence factors, 3 genes showed a significantly
different distribution. PapGII and tcpC were less frequent in GT
isolates than in UTI isolates (56% vs 78% and 7.6% vs 28%,
respectively), whereas ibeA was far more prevalent in GT isolates
than in UTI isolates (27% vs 2.7%) (Table 1). The mean
aggregate virulence score, calculated as the mean number of
unique virulence factors per strain, was nearly identical in UTI
and GT isolates (Table 1). In the GT subgroup, patients with
EOI were far younger than those with LOI (median age, 2 vs 24
days; P , .001) and the median term at birth was higher (median, 32.6 vs 30.0 weeks), meaning that postconception age was
not significantly different between the EOI and LOI GT subpopulations (median, 33.0 vs 33.4 weeks,). Several significant
differences emerged between the 2 subpopulations. Hemolysin
(hlyC) was significantly more common for LOI versus EOI (46%
vs 0%), whereas salmochelin (iroN) was more prevalent in EOI
(84% vs 38%; P 5 .02). Moreover, several plasmidic traits (cva,
etsC, iss, ompTp, and hlyF) occurred significantly more frequently in subject with EOI than in those with LOI, and so was
the prevalence of the CVP region (61.5% vs 15.3%; P 5 .01).
RESULTS
DISCUSSION
During the study period, 127 cases of E. coli bacteremia were
diagnosed in infants aged ,3 months. Twenty-seven cases were
excluded (8 cases of peritonitis, 3 cases of enterocolitis, 6 cases of
congenital gastrointestinal tract abnormalities, 4 cases of
diaphragmatic hernia, 2 catheter-related infections, and 4 UTIs
in patients with major urinary tract abnormalities). Therefore,
100 patients (74 with UTI and 26 with GT) met our case definitions (Table 1). GT isolates were equally distributed between
subjects with EOI and those with LOI. Infants with UTI were
significantly older than infants with GT (median age, 40 vs 4
days), and they also had a significantly higher ratio of malefemale sex (89% vs 42%) and median term at birth (39 vs 33
weeks). All but 9 of the infants had undergone lumbar puncture.
Three infants (2 with UTI and 1 with GT) had E. coli meningitis.
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Although the infants with GT bacteremia were markedly
younger than those with UTI bacteremia, and although they had
shorter gestations, no difference in the phylogenetic distribution
was found between GT and UTI isolates. Virulence groups B2
and D comprised nine-tenths of the strains. The mean aggregate
virulence score was also similar for GT and UTI isolates.
Therefore, GT isolates from infants with wall-bowel integrity
appear to be as virulent as those that cause urosepsis, and the
impaired innate immunity in young infants does not seem to
favor GT isolates that lack virulence factors or that belong to the
low-virulence phylogroups A and B1.
Our comparison of virulence determinants between UTI and
GT isolates delineates the pathogenic role of tcpC and ibeA in
Figure 1. Phylogenetic tree, rooted on Escherichia fergusonii, of Achtman's sequence types (STs) encountered among 100 E. coli strains causing
bacteremia in children aged ,3 months. The tree was constructed from the multilocus sequence typing results inferred from 7 housekeeping gene
sequences (3421 letters) by using the neighbor-joining algorithm. Bootstrap confidence values for each node of the tree were calculated over 100
replicate trees (only bootstrap values .80% are indicated). Fully sequenced reference strains indicated in boldface font are included in the tree to
distinguish the major phylogenetic groups (group A, K - 12; group B1, IAI1 and SE11; group B2, CFT073, 536, S88, and ED1a; and group D, UMN026 and
IAI39) and to classify our clinical isolates among them. The phylogenetic groups thus inferred from the MLST tree topology are indicated by large ovals.
A strain number of the Escherichia coli Reference (ECOR) collection is indicated after the ST number if relevant. ST complexes (STcs) are indicated. For
each ST or STc, the O antigens encountered are listed, thus defining sequence O-types by the combination of an ST or STc with an O serogroup. For each
sequence O-type, the number of isolates in the urinary tract infection (UTI) and gut translocation (GT) subgroups is indicated in brackets. OND, O antigen
not determined.
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this clinical setting. TcpC is a newly described virulence factor
that subverts innate immunity by interfering directly with Tolllike receptor functions and has been implicated experimentally
in uropathogenicity [8]. TcpC was significantly less prevalent in
our GT isolates than in our urosepsis isolates (7.6% vs 28%) and
was not more common than has reported elsewhere among fecal
isolates [8]. Thus, TcpC does not appear to be required for
neonatal bacteremia associated with GT, possibly because constitutive expression of TLR4 is lower in preterm infants than
in term newborns [11]. TcpC may therefore not confer an
advantage to ExPEC in preterm infants.
ibeA was initially described as being involved in the invasion
of brain microvascular endothelial cells. The prevalence of ibeApositive strains in our GT strain collection (27%) was not
markedly different from that described elsewhere in neonatal
meningitis isolates (33–38%) [4, 12], although only 1 of our
patients had meningitis. In contrast, ibeA occurred surprisingly
infrequently in our urosepsis strains (2.7%). Although these
results appear to point to a role of ibeA in GT, 2 recent findings
must be taken into account. First, Cortes et al [13] showed that
ibeA may play a major role in type I fimbriae regulation, leading
to permanent synthesis of the fimbriae. Thus, ibeA may cause
permanent adhesion to bladder epithelial cells and thereby
reduce the capacity of E. coli to reach the kidneys. Second,
Homeier et al [14] obtained evidence that the locus containing
ibeA is an ancestral part of group B2 and that 50% of B2 strains
causing UTI have lost this locus, suggesting that the urinary tract
may exert negative selective pressure on ibeA. The very low
prevalence of ibeA (2%) in our pyelonephritis isolates, together
with the findings of Cortes and colleagues and of Homeier and
colleagues, suggests that ibeA is incompatible with upper UTI,
rather than playing a role in GT. The relatively high rate of ibeApositive isolates among fecal B2 strains observed by Homeier
and colleagues (29%) and by ourselves (31%; data not shown),
together with the very strong negative association between ibeA
and the major pyelonephritis adhesin PapGII, supports this
hypothesis [4, 12].
Hek is the only ExPEC protein known to mediate adherence
to and invasion of gastrointestinal cells [3]. However, this factor
may also play a role in adherence to uroepithelial cells [3]. This
is supported by our results, because we observed a similar
prevalence of Hek in GT and UTI isolates (23% vs 28%).
Our third major finding concerns the difference between EOI
and LOI GT isolates. Soto et al [15] reported that the only
apparent difference between EOI and LOI isolates was a higher
prevalence of ibeA in the former, but we found no such difference. This discrepancy may be due to the fact that UTI represented 40% of cases of LOI in Soto and colleagues’ study. We
found no evidence of a different role of ibeA in EOI and LOI GT
isolates.
In contrast, we found that the CVP region and hlyC were
differently distributed between EOI and LOI GT isolates (table
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1). This difference was not related to different host susceptibility,
because the infants had similar postconceptional ages. In EOI, E.
coli has to circumvent the innate immune system, which keeps
the amniotic cavity sterile, including cervical mucus, the chorioamniotic membranes, and the antibacterial properties of
amniotic fluid. The CVP region—more prevalent in our EOI
isolates—includes numerous putative virulence factors and
genes of unknown function that may help to subvert these
innate defenses. Additional studies are needed to determine the
roles of CVP-region genes in EOI, particularly because they may
offer new vaccine targets for EOI and new markers of highly
virulent strains present in the birth canal.
Finally, the higher prevalence of a-hemolysin in LOI than in
EOI may be associated with maturation of the gastrointestinal
barrier. Of note, a-hemolysin has been implicated in renal epithelial translocation [16], but it remains to be shown whether it
is preferentially involved in LOI.
This is, to our knowledge, the first large molecular study of E.
coli strains recovered from young bacteremic infants with meticulous medical records clearly identifying the portal of entry
and patient status. We identified several genetic determinants
differentiating urosepsis isolates, EOI GT isolates, and LOI GT
isolates. Although gene detection by PCR may not perfectly
reflect the presence of the entire gene and its expression, our
work represents a first step towards defining 3 different E. coli
pathotypes causing bacteremia in young infants and may serve
to identify E. coli strains at high or low risk of bacteremia in
neonates.
Acknowledgments
Platform Genotyping of Pathogens and Public Health acknowledges
support from Institut de Veille Sanitaire (Saint-Maurice, France).
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