INFECTION AND IMMUNITY, May 2000, p.

30403047
0019-9567/00/$04.000
Copyright 2000, American Society for Microbiology. All Rights Reserved.

Vol. 68, No. 5

Molecular Epidemiological and Phylogenetic Associations of Two

Novel Putative Virulence Genes, iha and iroNE. coli, among
Escherichia coli Isolates from Patients with Urosepsis
JAMES R. JOHNSON,1* THOMAS A. RUSSO,2 PHILLIP I. TARR,3 ULRIKE CARLINO,2
SIMA S. BILGE,3 JAMES C. VARY, JR.,3 AND ADAM L. STELL1
Medical Service, VA Medical Center, and Department of Medicine, University of Minnesota, Minneapolis, Minnesota1;
Medical Service, VA Medical Center, and Department of Medicine and Center for Microbial Pathogenesis,
State University of New York at Buffalo, Buffalo, New York2; and Division of Gastroenterology,
Childrens Hospital and Regional Medical Center, and Department of Pediatrics,
University of Washington School of Medicine, Seattle, Washington3

Two novel putative Escherichia coli virulence genes, iha and iroN from E. coli (iroNE. coli), were detected in 55
and 39%, respectively, of 67 E. coli isolates from patients with urosepsis. iha and iroNE. coli exhibited divergent
associations with other putative virulence genes, phylogenetic markers, host characteristics, and antimicrobial
resistance.

gene) and allele II of papG (the P fimbrial digalactoside-specific adhesin gene) (8, 16, 17). Consistent with the hypothesis
that iha is a PVF in ExPEC, probes derived from PAI regions
immediately adjacent to iha as it occurs in strain CFT073
hybridized significantly more frequent with UTI or bacteremia
isolates of E. coli than with commensal E. coli (8).
iroNE. coli, a novel catechole siderophore receptor which
exhibits increased expression in urine, was recently identified
in archetypal ExPEC strain CP9 as part of a PAI which also
includes one of this strains two pap operons (i.e., the pap
operon containing the F14 papA allele and papG allele III),
cnf1 (named for cytotoxic necrotizing factor 1), hly, and foc
(F1C fimbriae) (16, 23). iroNE. coli, like iha, was also found by
probe hybridization to be more prevalent among E. coli isolates from patients with UTI or bacteremia than among commensal strains, consistent with its being a VF in extraintestinal
infections (23).
In the present study, we used PCR- and probe hybridizationbased gene detection to determine the prevalence of iha and
iroNE. coli within a well-characterized collection of E. coli blood
isolates from patients with urosepsis. By comparing newly determined results for iha and iroNE. coli with previously determined extended PVF genotypes (15), O:K:H serotypes (13),
carboxylesterase B electrophoretic types (which reflect membership in virulence-associated phylogenetic group B2 versus
in other phylogenetic groups) (11), antimicrobial resistance
profiles (12), and host compromise data (12), we were able to
define and compare the associations of iha and iroNE. coli with
these other factors. We present data demonstrating that iha
and iroNE. coli exhibit widely divergent associations with all of
the above factors and are not consistently linked even with
PVFs from their own presumed PAIs of origin.
Strains. The clinical collection studied comprised 67 wellcharacterized blood culture isolates of E. coli collected from
adults with urosepsis in Seattle, Wash., in the mid-1980s (12).
The status of these strains with respect to multiple characteristics has been reported previously (1013, 15). (Eight strains
from the 75-member collection which exhibited amplification
fingerprints inconsistent with their putative O:K:H serotype
and carboxylesterase B type in experiments not yet published

The virulent strains of Escherichia coli that cause urinary

tract infections (UTIs) and other extraintestinal infections in
humans (i.e., extraintestinal pathogenic E. coli [ExPEC]) owe
their pathogenic potential largely to the presence of specialized virulence factors (VFs) which are absent from commensal
members of the species and which allow ExPEC strains to
colonize host mucosal surfaces, injure and invade host tissues,
foil host defense mechanisms, and incite an injurious host
inflammatory response (4, 5, 9, 20). Currently recognized putative VFs (PVFs) of ExPEC include adhesins, siderophores,
toxins, protectins, and invasins, some of which are encoded on
pathogenicity-associated islands (PAIs) (2, 7, 8, 17, 24, 26).
PAIs are large blocks of established or suspected virulence
genes that are inserted into the E. coli genome (often at tRNA
loci) and which may provide a mechanisms for coordinate
horizontal transfer of virulence genes between lineages within
E. coli and even between species (3, 7, 21, 24, 25).
Two recently described PAI-linked PVF genes of ExPEC,
iha and iroN from E. coli (iroNE. coli), are of interest both in
their own right and because of their potential utility as markers
for their respective PAIs of origin (23, 27). iha, an novel nonhemagglutinating adhesin which in vitro confers HeLa cell
adherence capability to (nonadhering) E. coli K-12, was first
identified as part of a tellurite resistance-associated PAI
(termed TAI, the tellurite resistance-adherence-conferring island) from an E. coli O157:H7 isolate from a patient with
hemorrhagic colitis (27). iha exhibits nearly perfect sequence
identity with the sequenced portion of an open reading frame
(ORF) of unknown function (ORF R4) from a PAI in archetypal ExPEC strain CFT073 (8, 17). In addition to the iha
homologue and multiple other ORFs of unknown significance,
this PAI from strain CFT073 also contains an hly (hemolysin)
operon and one of the strains two pap operons (named for
pilus associated with pyelonephritis; P fimbriae), which includes the F7-2 allele of papA (the P fimbrial structural subunit

* Corresponding author. Mailing address: Infectious Diseases

(111F), Minneapolis VA Medical Center, One Veterans Dr., Minneapolis, MN 55417. Phone: (612) 725-2000, ext. 4185. Fax: (612) 7252273. E-mail: johns007@tc.umn.edu.
3040

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Received 29 December 1999/Returned for modification 10 February 2000/Accepted 23 February 2000

VOL. 68, 2000

3041

present study (Table 2), even though in strain CFT073 malX

occurs on the same PAI as iha and is separated from iha by 2
kb (8). iha occurred in seven strains that lacked the PAI
marker, whereas the PAI marker occurred in 19 strains that
lacked iha (Table 1). Similarly, despite its association with
papG allele III, iroNE. coli occurred in four pap-negative strains
and in 19 strains that contained papG allele II rather than
papG allele III, whereas papG allele III occurred in one strain
without iroNE. coli (Tables 1 and 2). An analogous discordance
was also noted for both iha and iroNE. coli with respect to hly
(Tables 1 and 2).
Not only were iha and iroNE. coli inconsistently associated
with PVFs from their respective PAIs of origin, but they exhibited divergent associations with other PVFs. iha and
iroNE. coli were concordantly positively associated only with
focG and hlyA, and exhibited no concordant negative associations (Table 3). In contrast, they exhibited discordant associations with 20 of the remaining PVFs (Table 4). (Of note,
whereas iha was strongly negatively associated with plasmid iut
[aerobactin] and cvaC [colicin V], iroNE. coli was strongly positively associated with plasmid aerobactin and exhibited a trend
toward an association with cvaC [Tables 2 and 3].) Thus, iha
and iroNE. coli differed more often than they agreed with respect to associations with other PVF genes (Table 3).
Associations with papA alleles. iha and iroNE. coli differed
substantially with respect to their associations with individual
papA alleles (Table 4). iha was significantly associated with the
F7-2 allele, which is present in the PAI of strain CFT073 that
contains iha, and with the F10 and F16 alleles, whereas it was
significantly negatively associated with the F11 allele (Table 4).
In contrast, iroNE. coli was positively associated with the F14
allele (P 0.029), which is present in the iroNE. coli-containing
PAI from strain CP9. iha and iroNE. coli exhibited (nonsignificant) opposing associative trends with all the remaining papA
alleles except F13 (Table 4).
Phylogenetic distribution of iha and iroNE. coli. Although iha
and iroNE. coli were both somewhat more prevalent among
carboxylesterase B2 strains, they exhibited divergent patterns
of distribution with respect to individual O:K:H serotypes (Table 1). In contrast to the more homogeneously distributed iha,
iroNE. coli was sporadically distributed, occurring in many different serotypes but rarely in all members of any one serotype
(Table 1). Although both PVFs occurred in a similar proportion of the 14 most prevalent O:K:H serotypes (i.e., 10 of 14 for
iha versus 9 of 14 for iroNE. coli), iha satisfied criteria for
homogeneous distribution (as defined above [phylogenetic
analysis]) in all 14 serotypes, as compared with only seven for
iroNE. coli (P 0.02, McNemars test) (Table 1).
Among the B2 strains, iha was almost universally prevalent
except among O1:K1:H7 and O2:K1:H7 strains, from which it
was notably absent (P 0.001 versus other B2 strains). In
contrast, although iroNE. coli was also absent from the five
O1:K1:H7 strains, it occurred in three of the five (iha-negative)
O2:K1:H7 strains and was absent from all four (iha-positive)
O75 strains (Table 1). Likewise, among the B1 strains, whereas
iha was largely confined to serotypes O7:K1, O15:K52, O25:
K2, and serogroup O157 (P 0.001 versus other B1 strains),
iroNE. coli was confined to strains of serogroups O8 and O9
(P 0.001 versus other B1 strains), which resulted in a significant negative association of iha with iroNE. coli among B1
strains (P 0.004) (Table 1).
Host compromise and antimicrobial resistance. iha and
iroNE. coli differed with respect to their associations (or lack
thereof) with host compromise and antimicrobial resistance.
iha was consistently more prevalent among strains from noncompromised hosts than those from compromised hosts, with

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were excluded from the present study, leaving 67 evaluable

strains as studied herein.)
Molecular detection of iha and iroNE. coli. iha and iroNE. coli
were each detected in duplicate both by PCR and by dot blot
hybridization. Primers for iha, which were based on GenBank
accession no. AF126104 (27), were 5-CTGGCGGAGGCTC
TGAGATCA-3 (IHA-F; forward) and 5-TCCTTAAGCTC
CCGCGGCTGA-3 (IHA-R; reverse) (827-bp product). Primers for iroNE. coli, which were based on GenBank accession no.
AF135597 (23), were 5-AAGTCAAAGCAGGGGTTGCCC
G-3 (IRONEC-F; forward) and 5-GACGCCGACATTAAG
ACGCAG-3 (IRONEC-R; reverse) (665-bp product). Probes
were generated and PCR labeled as previously described (14)
using the same primers as for PCR-based gene detection. Amplification and blotting procedures were as previously described (14, 15). Appropriate positive and negative controls
were included in each assay. Discrepancies between duplicate
blot or PCR determinations were investigated with additional
replicates as needed.
Phylogenetic analysis of PVF distribution. Strains were
sorted according to carboxylesterase B electrophoretic type
(i.e., B2 or B1) and then by O:K:H serotype within each carboxylesterase B type. Serotypes with two or more representatives in the study population were considered to exhibit homogeneity with respect to a particular PVF if the PVF was present
in or absent from all representatives of the serotype or was
present in all representatives but one (if three or more representatives total).
Statistical analyses. Comparisons of proportions were
tested using Fishers exact test. Comparisons of prevalence
within the same population were tested using McNemars test
(6). Comparisons of antibiotic resistance scores and host compromise scores were tested using the Mann-Whitney U test.
The criteria for the different categories of statistical significance follow: P 0.10, indifferent association; P 0.10, borderline significant trend; P 0.05, possible statistical significance, and P 0.01, statistical significance. Associations of iha
and iroNE. coli with another PVF were considered to agree
when they were in the same direction and exhibited P values of
0.10, to somewhat disagree (regardless of direction) when
one exhibited a P value of 0.05 and the other exhibited a P
value of 0.10, and to strongly disagree when the associations
were in opposite directions and both exhibited P values of
0.10.
Prevalence of iha and iroNE. coli. iha was detected in 37
(55%) and iroNE. coli in 26 (39%) of the 67 urosepsis isolates
(0.05 P 0.10, McNemars test) (Table 1). Probe and PCR
yielded identical results for each gene (not shown). Overall, iha
and iroNE. coli were indifferently associated with one another
(P 0.10).
Associations with other PVFs. iha and iroNE. coli each exhibited positive, negative, and indifferent associations with diverse
other PVFs (Table 2). Consistent with the occurrence of iha in
archetypal uropathogenic strain CFT073 on a PAI that also
contains papG allele II and hly (8, 15), iha was strongly associated with papG allele II (and all other pap elements except
papG allele III) and with hlyA (Table 2). Similarly, consistent
with the discovery of iroNE. coli in archetypal uropathogenic
strain CP9 on a PAI that also contains papG allele III, hly, cnf,
and foc, iroNE. coli was positively associated with each of these
traits, despite not being significantly associated with other pap
elements (Table 2).
However, associations of iha and iroNE. coli with other genes
from their respective PAIs in strains CFT073 and CP9 were
not categorical. For example, iha exhibited only a marginal
statistical association with malX, the PAI marker used in the

NOTES

O2:K5:H1
O2:K7:H
O2:K7:H
O2:K7:H
O2:K7:H1

O4:K12:H1
O4:K12:H
R:K12:H1

B2
B2
B2
B2
B2

NAc
B2
B2

B2
B2
B2
B2
B2
B2
B2
B2
B2
B2

B2
B2

B2
B2

B2

B2
B2
B2

B2

B2

B2

V27
H19
V6
V24
H1

V31
PM8
V7

H26
V5
U3
PM4
PM5
2H4
2H24
V1
V3
V16

V19
V22

H3
U5

U7

H9
U4
PM2

H7

2H25

H5

II

O18ac:K5:H F8, F10

II

II
II

III

II
III

II
II

II
II
II
II
II
II
II
II
II
II

II
II
II

II
II,III
II
II
II

II
II
II
II
II

II
II
II
II
II

III

F7-1

O16:K1:H

Adhesin

Toxin

Siderophore

and other genes among 67 urosepsis isolatesa

Protectin or invasin

d
d
d
d
d
d
d
d
d
d

kpsMT kpsMT
G
sfa/
afa/
K1 K5 rfc cvaC traT ibeA
sfaS focG
iha bmaE gafD hlyA cnf1 cdtB iroNE. coli fyuA iutA
II
III
allele(s) focDE
draBC

O18ac:K1:H7 F10

F9, F12, F48

F12, F48
NEGe

F48

O6:K?:H

O12:K1:H6
O12:K1:H6
R:K1:H6

F11
F10, F14

F10, F13
F10, F13

O6:K5:H
O6:K5:H

O6:K53:H7
O6:K53:H7

F7-2
F7-2
F7-2
F7-2
F7-2
F7-2
F7-2
F7-2
F7-2
F7-2

F10, F11
F10, F11
F10, F11, F16

F10, F14
F10, F14
F10, F14
F7-1, F10
F10, F12, F15

F11
F11
F11
F11
F11

F11
F11
F11
F11
F11

F-type

O6:K2:H1
O6:K2:H1
O6:K2:H1
O6:K2:H1
O6:K2:H1
O6:K2:H1
O6:K2:H1
O6:H1
O6:K2:H?
R:K2:H1

O2:K1:H7
O2:K1:H7
O2:K1:H7
O2:K1:H7
R:K1:H7

B2
B2
B2
B2
B2

H15
H25
PM6
2P6
H35

O1:K1:H7
O1:K1:H7
O1:K1:H7
O1:K1:H7
O1:K1:H7

B2
B2
B2
B2
B2

H16
H38
V15
V23
2H18

O:K:H
serotype

coli

Miscellaneous
(PAI)

NOTES

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Strain CBTb

TABLE 1. Prevalence of iha and iroNE.

3042
INFECT. IMMUN.

B2
B2
B2

B2

B1

B1
B1
B1

B1
B1

B1

B1

B1
B1

B1

B1

B1
B1

B1

B1
B1

B1

B1

B1

B1

B1

B1

V21
2H19
V8

PM3

V14

V20
V29
PM7

H27
U6

U1

V28

V9
PM9

V26

V32

2H17
2P9

V11

2H16
H8

H17

PM1

V10

V12

H2

H18

O77:K54:H18 NEG

O84:H

F16

NEG

NEG

NEG

O157:K52:H45 F16

O157:H32

O64:H21

O25:K16:H4

F16
F16

II

II

II
II

O25:K2:H2
O25:K2:H2

O17:K53:H18 NEG

F16
F16

O15:K52:H1
O15:K52:H1

II
II

III

II

II

II
II
II

II

II

II

F14

O15,O40:K

NEG
NEG

O9:K34:H
O9:K34:H

F11

NEG

O8:K:H

O9:K36:H19

NEG

F11
NEG

O8:K27:H
O8:K27:H

O8:K44:H9

F10
F40
F10

O7:K1:H
O7:K1:H
O7:H

O2:K5:H

F10, F48

F10

O75:K:H

F10
F8, F10
F10

O75:K5:H5
O75:K5:H5
O75:K5:H

O21:K100:H5 F7-2

d
d

NOTES

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The presence () or absence () of the indicated gene in isolates is shown. The iha and iroN data are shown in bold type for emphasis. Data are adapted from reference 15, with permission.
b
CBT, carboxylesterase B type.
c
NA, not applicable.
d
Negative by PCR but positive by probe for kpsMT II.
e
NEG, no F-type adhesin.

B2

2H21

VOL. 68, 2000

3043

3044

NOTES

INFECT. IMMUN.
TABLE 2. Distribution of other PVFs according to the presence or absence of iha and iroNE.

coli

Prevalence (no. [%]) of associated PVF

Associated PVFa

iha
Present
(n 37)

Absent
(n 30)

55 (82)
53 (79)
49 (73)
5 (7)
18 (27)
3 (4)
13 (19)
4 (6)
4 (6)
1 (1)
29 (43)
12 (18)
6 (9)
63 (94)
52 (77)

35 (95)
34 (92)
34 (92)
1 (5)
13 (35)
0
12 (33)
4 (11)
0
0
22 (59)
6 (16)
5 (14)
37 (100)
37 (100)

20 (67)
19 (63)
15 (50)
4 (13)
5 (17)
3 (10)
1 (3)
0
4 (13)
1 (3)
7 (23)
6 (20)
1 (3)
26 (87)
15 (50)

13 (19)
39 (58)
56 (84)
19 (28)
12 (18)
25 (33)
1 (1)
3 (4)
4 (6)
9 (13)
44 (66)
48 (72)
29 (39)

1 (3)
36 (97)
36 (97)
6 (16)
11 (30)
19 (51)
0
2 (5)
1 (3)
0
21 (57)
30 (81)
13 (35)

12 (40)
3 (10)
20 (67)
13 (43)
1 (3)
6 (20)
1 (3)
1 (13)
3 (10)
9 (30)
23 (77)
18 (60)
13 (43)

iroNE. coli
Present
(n 26)

P valueb

0.004
0.006
0.001
(0.08)
0.004
(0.036)
0.006
0.036
0.001
(0.001)
0.001
0.002
(0.03)
0.008
0.01

(0.001)
0.10

22 (85)
22 (85)
19 (73)
4 (15)
18 (69)
3 (12)
13 (50)
0
2 (8)
1 (4)
17 (65)
10 (38)
4 (15)
26 (100)
21 (81)
8 (31)
13 (50)
21 (81)
5 (19)
6 (23)
10 (38)
0
2 (8)
3 (12)
8 (31)
16 (62)
21 (81)
NAc

Absent
(n 41)

P value

33 (80)
31 (76)
30 (73)
1 (2)
0
0
0
4 (10)
2 (5)
0
12 (29)
2 (5)
2 (5)
37 (90)
31 (76)

0.07
0.001
0.054
0.001

0.005
0.001

5 (12)
26 (63)
35 (85)
14 (34)
6 (15)
15 (37)
1 (2)
1 (2)
1 (2)
1 (2)
28 (68)
27 (66)
NA

0.002

a
As determined by PCR only (K1 and K5), probe only (plasmid aerobactin and kpsMT II), or both PCR and probe (all other PVFs). K2 represents isolates that
were blot positive but PCR negative for kpsMT II. K5 represents isolates with non-K1, non-K2 group II capsules. Plasmid aerobactin status was determined previously
(12). Chromosomal aerobactin status was inferred from results for iutA and plasmid aerobactin. No strain had nfaE; all strains had fimH. Results for papC and papEF
were similar to those for papAH and papG.
b
P values (by Fishers exact test) shown only where 0.10. Parentheses indicate negative association of iha with particular PVF.
c
NA, not applicable (comparison with self).

the strength of the association depending on the type of host

compromise (Table 5). In contrast, iroNE. coli was more prevalent among strains from compromised hosts (Table 5). Host
compromise scores were significantly lower among iha-positive
strains (mean, 0.8 [range, 02]) than among iha-negative
strains (mean, 1.4 [range, 03]; P 0.002) but did not differ
between iroNE. coli-positive and -negative strains (means of 1.0
and 1.1, respectively [range for both groups, 03]; P 0.10).

Among strains from noncompromised hosts, iha was always

somewhat or significantly more prevalent than was iroNE. coli,
whereas among strains from compromised hosts, iha and
iroNE. coli were similarly prevalent (Table 5).
Resistance to one or more antimicrobial agent was less common among iha-positive strains than among iha-negative
strains (38 versus 67%; P 0.03), whereas it was comparably
prevalent among iroNE. coli-positive and -negative strains (46

TABLE 3. Summary of agreement and disagreement between iha and iroNE.

Agree
(positive vs positiveb)

focG, hlyA

coli

with respect to associations with other PVFs:a

Somewhat disagree
Positive vs indifferentc

Indifferent vs positived

Negative vs indifferente

Strongly disagree
(Negative vs positive f)

papAH, papG allele II, fyuA, iutA,

kpsMT II, K2, K5, PAI

papG allele IIIg, sfa/foc, cnf1

bmaE, K1, traTh, plasmid iut

sfaSg,h, cvaC

a
Of the PVFs present in two or more strains each, iha and iroNE. coli were both indifferently associated with afaBC, cdtB, gafD, rfc, and ibeA but exhibited opposed
associative trends with all but rfc. PVFs which were present in 0, 1, or all 75 of the urosepsis isolates, including gafD, nfaE, fimH, and kpsMT III, were excluded from
the present analysis.
b
Positive associations of both iha and iroNE. coli with other PVFs.
c
Association with other PVFs: positive (iha), indifferent (iroNE. coli) (this also applies to papC papEF, and papG [not shown]).
d
Association with other PVFs: indifferent (iha), positive (iroNE. coli).
e
Association with other PVFs: negative (iha), indifferent (iroNE. coli).
f
Association with other PVFs: negative (iha), positive (iroNE. coli).
g
For iroNE. coli, 0.05 P 0.10.
h
For iha, 0.05 P 0.10.

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papAH
papG
Allele II
Allele III
sfa/foc
sfaS
focG
afa/draBC
bmaE
gafD
hlyA
cnf1
cdtB
fyuA
iutA
iut
Plasmid
Chromosomal
kpsMT II
K1
K2
K5
kpsMT III
rfc
ibeA
cvaC
traT
PAI marker
iroNE. coli

No.
(% of total)

VOL. 68, 2000

NOTES
TABLE 4. Distribution of papA alleles according to the presence or absence of iha and iroNE.

3045

coli

Prevalence (no. [%]) of associated papA allele

No.
(% of total)

F7-1
F7-2
F8
F9
F10
F11
F12
F13
F14
F15
F16
F48

2 (3)
11 (16)
2 (3)
1 (1)
18 (27)
15 (22)
3 (4)
2 (3)
6 (9)
1 (1)
8 (12)
4 (6)

iha

iroNE. coli

Present
(n 37)

Absent
(n 30)

2 (5)
10 (27)
2 (5)
1 (3)
15 (41)
1 (3)
3 (8)
2 (5)
4 (11)
1 (3)
8 (22)
3 (8)

0
1 (3)
0
0
3 (10)
14 (47)
0
0
2 (7)
0
0
1 (3)

P valueb

0.017
0.006
(0.001)c

0.007

Present
(n 26)

Absent
(n 41)

0
6 (23)
1 (4)
0
9 (35)
5 (19)
0
2 (8)
5 (19)
0
1 (4)
1 (4)

2 (5)
5 (12)
1 (2)
1 (2)
9 (22)
10 (24)
3 (7)
0
1 (2)
1 (2)
7 (17)
3 (7)

P value

0.029

a
As determined by PCR (16). papA alleles F7-1 through F16 correspond with the traditionally recognized P fimbrial F serotypes. F48 is a recently described novel
papA allele.
b
P values (by Fishers exact test) shown only where 0.10.
c
Parentheses indicate a negative association of iha with F11.

versus 54%; P 0.10). Similarly, antimicrobial resistance

scores were lower among iha-positive strains than among ihanegative strains (mean of 0.8 [range, 07] versus 3.0 [range,
010]; P 0.002) but were similar among iroNE. coli-positive
and -negative strains (mean of 2.0 [range, 010] versus 1.7
[range, 09]; P 0.10).
Implications of findings. That iha and iroNE. coli were both
significantly associated with other PVF genes from their respective PAIs in archetypal ExPEC strains CFT073 (iha) and
CP9 (iroNE. coli) suggests, as a first approximation, that iha and
iroNE. coli occur consistently on the same (or similar) PAIs
throughout the ExPEC population. Consequently, the striking
differences documented in the present study between iha and
TABLE 5. Distribution of iha and iroNE.

coli

iroNE. coli with respect to their individual associations with

other bacterial and host characteristics demonstrate that such
associations are likely to be specific to individual PAIs and/or
PVF genes rather than being common to all PAIs or to PAIlinked PVF genes in general. This implies that discovery that a
particular PVF of ExPEC is linked to a PAI is insufficient to
predict its associations with other bacterial and host characteristics, which instead can be discovered only through specific
study of the particular PVF itself or the individual PAI.
However, our findings also suggest that the concept of an
individual PAI as a discrete entity which can be tracked
through the ExPEC population may itself be illusory (1, 2, 7, 8,
23, 26). The associations observed in the present study of iha

according to the presence or absence of host compromise

Prevalence (%) of ihaa

Host compromise condition

Host condition
Present

Absent

Prevalence (%) of iroNE.

P valueb

Host condition
Present

Absent

48
50
54
69
38

33f
37g
35f
31f
39g

Medical illnessc
Diabetes mellitus
Cancer
Immunosuppression
Uremia

40
50
31
46
25

64f
56g
61f
57f
59g

Urinary tract abnormalityd

Upper urinary tract

47
13

62
56h

(0.018)

37
38

41
39h

Urinary tract instrumentation

25

65h

(0.009)

38

39h

Any of abovee

44

77f

(0.018)

42

32f

(0.076)
(0.065)

coli

P value

0.02

0.03

The denominator for prevalence values was the number of patients with or without the indicated compromising condition.
For comparisons between patients with versus patients without the indicated type of host compromise, P values (by Fishers exact test) shown only where 0.10.
(Parentheses indicate negative associations of iha with host compromise.)
c
Including diabetes mellitus, cancer, immunosuppression, and uremia.
d
Preexisting functional or anatomic abnormality of the urinary tract.
e
Any of the above conditions, i.e., medical illness or urinary tract abnormality or instrumentation.
f
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, P 0.02 (McNemars test). Among patients with the
indicated host compromise condition, P 0.10 for all comparisons of iha versus iroNE. coli.
g
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, 0.05 P 0.10 (McNemars test). Among patients with
the indicated host compromise condition, P 0.10 for all comparisons of iha versus iroNE. coli.
h
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, and P 0.05 (McNemars test). Among patients with the
indicated host compromise condition, P 0.10 for all comparisons of iha versus iroNE. coli.
b

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Associated
papA allelea

3046

NOTES

virulence genes, other explanations should be considered. Alternative mechanisms include coselection for the associated
traits (independent of these traits genetic relationship to one
another), and coselection for unmeasured traits that are genetically linked to the statistically associated putative virulence
genes. Confirmation of actual genetic linkage between putative
virulence genes would require direct genetic analyses. The
results of the present study can assist the design of such analyses by suggesting which linkages should be sought experimentally.
The significantly higher prevalence of iha found in the
present study among E. coli bacteremia isolates from noncompromised hosts than in those from compromised hosts suggests
that iha (or VFs linked to iha) may assist in overcoming host
defenses that are breached in patients with the types of compromise studied (12). In contrast, our observation that the
prevalence of iroNE. coli increased in the presence of host compromise suggests that unlike iha, iroNE. coli should be as good
a target for an anti-VF intervention among compromised hosts
as among noncompromised hosts. The absence of an association of iroNE. coli with antimicrobial resistance suggests that
iroNE. coli should also remain as effective a target for an anti-VF intervention among multidrug-resistant E. coli as among
susceptible strains, whereas iha, which was negatively associated with antimicrobial resistance, would be most useful as a
target among antimicrobial-susceptible strains.
It should be noted that the present study population represented a single geographical locale, a single clinical syndrome,
and a time period of several years in the mid-1980s. Analysis of
strains from other locales, clinical syndromes, and time periods
for iha and iroNE. coli would provide a valuable complement to
the present study.
This work was supported in part by VA Merit Review (J.R.J. and
T.A.R.); National Institutes of Health grants DK-47504 (J.R.J.), AI42059 (T.A.R.), and AI-38419 (P.I.T.); United States Department of
Agriculture grant 94-03953 (P.I.T.); and a National Cattlemens Beef
Association grant (P.I.T.).
Diana Owensby and Ann Emery helped prepare the manuscript.
REFERENCES
1. Bloch, C. A., and C. K. Rode. 1996. Pathogenicity island evaluation in Escherichia coli K1 by crossing with laboratory strain K-12. Infect. Immun. 64:
32183223.
2. Blum, G., M. Ott, A. Lischewski, A. Ritter, H. Imrich, H. Tschape, and J.
Hacker. 1994. Excision of large DNA regions termed pathogenicity islands
from tRNA-specific loci in the chromosome of an Escherichia coli wild-type
pathogen. Infect. Immun. 62:606614.
3. Boyd, E. F., and D. L. Hartl. 1998. Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J. Bacteriol. 180:11591165.
4. Donnenberg, M. S., and R. A. Welch. 1996. Virulence determinants of uropathogenic Escherichia coli. In H. L. T. Mobley and J. W. Warren (ed.),
Urinary tract infections: molecular pathogenesis and clinical management.
ASM Press, Washington, D.C.
5. Eisenstein, B. I., and G. W. Jones. 1988. The spectrum of infections and
pathogenic mechanisms of Escherichia coli. Adv. Intern. Med. 33:231252.
6. Fleiss, J. L. 1981. Statistical methods for rates and proportions. John Wiley
& Sons, New York, N.Y.
7. Groisman, E. A., and H. Ochman. 1996. Pathogenicity islands: bacterial
evolution in quantum leaps. Cell 87:791794.
8. Guyer, D. M., J.-S. Kao, and H. L. T. Mobley. 1998. Genomic analysis of a
pathogenicity island in uropathogenic Escherichia coli CFT073: distribution
of homologous sequences among isolates from patients with pyelonephritis,
cystitis, and catheter-associated bacteriuria and from fecal samples. Infect.
Immun. 66:44114417.
9. Johnson, J. R. 1991. Virulence factors in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80128.
10. Johnson, J. R. 1998. papG alleles among Escherichia coli strains causing
urosepsis: associations with other bacterial characteristics and host compromise. Infect. Immun. 66:45684571.
11. Johnson, J. R., P. H. Goullet, B. Picard, S. L. Moseley, P. L. Roberts, and
W. E. Stamm. 1991. Association of carboxylesterase B electrophoretic pat-

Downloaded from http://iai.asm.org/ on May 30, 2016 by guest

and iroNE. coli with other PVFs from their respective PAIs in
source strains CFT073 and CP9, although statistically significant, were not absolute, evidence which suggests that the corresponding PAIs may have undergone considerable recombination over time, with acquisition or deletion of various PVF
genes, or that each PVF has independently entered multiple
different PAIs. Specific evidence of the fluidity of PAIs was
found in the discordant occurrence of iha and the PAI marker
malX (Tables 1 and 2), genes which on their shared PAI in
strain CFT073 are located within 2 kb of one another (8).
Thus, it is likely that at the same time that PAIs are generating
diversity within the species by introducing new genetic material
into disparate lineages via horizontal transfer (7), PAIs themselves are undergoing diversification through deletion and insertion of PVF genes, some of which may be more mobile (or
labile) than are the PAIs themselves. PAIs thus may exist as
continuously evolving mosaic constructs, a phenomenon which
would be predicted to confound efforts to define a reliable
marker for the PAIs of ExPEC in general or for any particular
PAI. An example of this phenomenon may be the apparent
insertion of the group III capsule genes into the group II
capsule locus in strain CP9 (24). Similarly, TAI, the PAI on
which iha was first identified, was conserved in structure in
some, but not all, diarrheagenic E. coli tested (27).
The respective patterns of phylogenetic distribution of iha
and iroNE. coli suggest divergent evolutionary histories and
mechanisms of horizontal mobility, iha was present in almost
all strains of carboxylesterase B type B2 except for those of
serotypes O1:K1:H7 and O2:K1:H7 and was also highly prevalent in selected lineages among the non-B2 strains. This is
consistent with early acquisition of iha by the B2 phylogenetic
group soon after the differentiation of B2 strains from other E.
coli (18) but after the O1:K1:H7 and O2:K1:H7 clonal group
(which constitutes a discrete evolutionary cluster within the B2
group [19]) had split off from a common B2 ancestor. In this
scenario, the appearance of iha among non-B2 strains would
require the independent acquisition of iha by non-B2 lineages
through horizontal transfer either from a B2 source or from an
unknown external source. As an alternative scenario, iha could
have entered E. coli prior to the differentiation of the B2
phylogenetic group and subsequently have been deleted from
the ancestor of the O1:K1:H7 and O2:K1:H7 branch of the B2
group after the differentiation of this branch from other B2
strains. In this scenario, iha could have been inherited vertically from a common ancestor by both B2 and certain non-B2
strains, for example, those of closely related phylogenetic
group D (18).
In contrast to iha, the sporadic appearance of iroNE. coli in
many different serotypes strongly suggests either multiple horizontal acquisition events or multiple scattered deletions of
iroNE. coli within otherwise iroNE. coli-positive lineages. Although in strain CP9 iroNE. coli occurs on a genomic PAI (23),
the possibility of plasmid-associated transfer of iroNE. coli in
some strains was suggested in the present study by the striking
statistical association of iroNE. coli with cvaC (colicin V), which
occurs on large conjugative plasmids (2830). Precedent in
ExPEC for a siderophore system which occurs variously on
colicin V plasmids and on the genome is provided by the
aerobactin system, which has been postulated to represent an
extinct transposon (22, 28). The finding that in strain CP9
iroNE. coli is flanked by IS1230 sequences (23) suggests the
possibility of independent mobility of iroNE. coli, which would
be consistent with exchange of iroNE. coli between genome and
plasmids.
Although direct genetic linkage is a plausible explanation for
the observed statistical associations between different putative

INFECT. IMMUN.

VOL. 68, 2000

12.

13.

14.
15.
16.

18.
19.
20.
21.

Editor: E. I. Tuomanen

22.
23.
24.

25.

26.
27.

28.
29.
30.

3047

F. Scheutz, and J. Hacker. 1995. tRNA genes and pathogenicity islands:

influence on virulence and metabolic properties of uropathogenic Escherichia coli. Mol. Microbiol. 17:109121.
Roberts, M., S. ParthaSarathy, M. K. L. Lam-Po-Tang, and P. H. Williams.
1986. The aerobactin iron uptake system in enteropathogenic Escherichia
coli: evidence for an extinct transposon. FEMS Microbiol. Lett. 37:215219.
Russo, T. A., U. B. Carlino, A. Mong, and S. T. Jodush. 1999. Identification
of genes in an extraintestinal isolate of Escherichia coli with increased expression after exposure to human urine. Infect. Immun. 67:53065614.
Russo, T. A., S. Wenderoth, U. B. Carlino, J. M. Merrick, and A. J. Lesse.
1998. Identification, genomic organization, and analysis of the group III
capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5). J. Bacteriol. 180:338
349.
Schubert, S., A. Rakin, H. Karch, E. Carniel, and J. Heesemann. 1998.
Prevalence of the high-pathogenicity island of Yersinia species among
Escherichia coli strains that are pathogenic to humans. Infect. Immun 66:
480485.
Swenson, D. L., N. O. Bukanov, D. E. Berg, and R. A. Welch. 1996. Two
pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning
and sample sequencing. Infect. Immun. 64:37363743.
Tarr, P. I., S. S. Bilge, J. C. Vary, Jr., S. Jelacic, R. L. Habeeb, T. R. Ward,
M. R. Baylor, and T. E. Besser. 2000. Iha: a novel Escherichia coli O157:H7
adherence-conferring molecule encoded on a chromosomal region of conserved structure. Infect. Immun. 68:14001407.
Waters, V. L., and J. H. Crosa. 1991. Colicin V virulence plasmids. Microbiol. Rev. 55:437450.
Williams, P. H. 1979. Novel iron uptake system specified by ColV plasmids:
an important component in the virulence of invasive strains of Escherichia
coli. Infect. Immun. 26:925932.
Williams, P. H., and P. J. Warner. 1980. ColV plasmid-mediated, colicin
V-independent iron uptake system of invasive strains of Escherichia coli.
Infect. Immun. 29:1116.

Downloaded from http://iai.asm.org/ on May 30, 2016 by guest

17.

tern with presence and expression of urovirulence factor determinants and

antimicrobial resistance among strains of Escherichia coli causing urosepsis.
Infect. Immun. 59:23112315.
Johnson, J. R., S. Moseley, P. Roberts, and W. E. Stamm. 1988. Aerobactin
and other virulence factor genes among strains of Escherichia coli causing
urosepsis: association with patient characteristics. Infect. Immun. 56:405
412.
Johnson, J. R., I. Orskov, F. Orskov, P. Goullet, B. Picard, S. L. Moseley,
P. L. Roberts, and W. E. Stamm. 1994. O, K, and H antigens predict
virulence factors, carboxylesterase B pattern, antimicrobial resistance, and
host compromise among Escherichia coli strains causing urosepsis. J. Infect.
Dis. 169:119126.
Johnson, J. R., T. A. Russo, J. J. Brown, and A. Stapleton. 1998. papG alleles
of Escherichia coli strains causing first episode or recurrent acute cystitis in
adult women. J. Infect. Dis. 177:97101.
Johnson, J. R., and A. L. Stell. 2000. Extended virulence genotypes of
Escherichia coli strains from patients with urosepsis in relation to phylogeny
and host compromise. J. Infect. Dis. 181:261272.
Johnson, J. R., A. L. Stell, F. Scheutz, T. T. OBryan, T. A. Russo, U. B.
Carlino, C. C. Fasching, J. Kavle, L. Van Dijk, and W. Gaastra. 2000.
Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic
Escherichia coli using a novel multiplex PCR-based assay. Infect. Immun.
68:15871599.
Kao, J., D. M. Stucker, J. W. Warren, and H. L. T. Mobley. 1997. Pathogenicity island sequences of pyelonephritogenic Escherichia coli CFT073 are
associated with virulent uropathogenic strains. Infect. Immun. 65:28122820.
Lecointre, G., L. Rachdi, P. Darlu, and E. Denamur. 1998. Escherichia coli
molecular phylogeny using the incongruence length difference test. Mol.
Biol. Evol. 15:16851695.
Ochman, H., and R. K. Selander. 1984. Evidence for clonal population
structure in Escherichia coli. Proc. Natl. Acad. Sci. USA 82:198201.
Orskov, I., and F. Orskov. 1985. Escherichia coli in extra-intestinal infections.
J. Hyg. 95:551575.
Ritter, A., G. Blum, L. Emody, M. Kerenyi, A. Bock, B. Neuhierl, W. Rabsch,

NOTES