Vol. 61, No. 6

INFEcrION AND IMMUNITY, June 1993, p. 2453-2461

0019-9567/93/062453-09$02.00/0 Copyright X 1993, American Society for Microbiology

Structure and Copy Number Analyses of pap-, sfa-, and afaRelated Gene Clusters in F165-Positive Bovine and Porcine Escherichia coli Isolates SOURINDRA N. MAITI, JOSEE HAREL,* AND JOHN M. FAIRBROTHER GREMIP, Faculte de Medecine Veterinaire, Universite de Montreal, Saint-IHyacinthe, Quebec, Canada J2S 7C6 Received 16 November 1992/Accepted 22 March 1993

Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E. coil fimbrial operonspap, sfa, and afal. Probes from different segments of thepap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E. coli isolates possessingpap- and sfa-, pap- and aJa-, orpap-related sequences. Only single copies of thepap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable. Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences. Gene clusters related to pap were structurally similar to thepap operon in certain isolates or theprs operon in others. sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon. afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52. These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototypepap operon andfoc operon of the P family and Sfa family, respectively. afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afal operon.

copies of pap-related sequences (2, 17, 39). These isolates express one, both, or neither of the P and Prs adhesins. S (Sfa) fimbrial adhesins, which are associated with E. coli strains causing newborn meningitis and urinary tract infection, mediate adhesion to ot-sialyl-0-2,3-Gal-containing receptors (14). The sfa operon, encoding S fimbrial adhesin, has been cloned and characterized (14). FlC and Sfr, which are devoid of any detectable receptor specificity, are genetically highly related to Sfa (37, 38). Although no detectable receptor for FlC is known, strains possessing these fimbriae adhere to buccal epithelial cells and to some renal tissues (1, 37). Urinary tract E. coli isolates expressing MRHA of human 0 erythrocytes in the absence of P fimbriae have been described as having X fimbriae (19, 23). The Dr family of adhesins, which bind to the Dr blood group antigen, includes the 075X adhesin and adhesins Afal and AfaIII. Genetic determinants encoding the 075X, AfaI, and AfaIII adhesins have been cloned, characterized, and found to be closely related to each other (22, 33). However, structural heterogeneity in the genetic determinant encoding the structural adhesin has been demonstrated among a group of E. coli clinical isolates expressing the X adhesin and possessing afaI-related sequences (19, 22). Fimbrial antigen complex F165 is found mostly on E. coli of serogroups 08, 09, 0101, 0115, and 0141, isolated from piglets and calves (11, 15) with septicemia or with various diseases for which diarrhea is the most prominent clinical sign. Most F165-positive E. coli isolates are nonenterotoxigenic, produce aerobactin, are resistant to the bactericidal effects of serum, do not produce verotoxin, and are negative for fimbrial antigens F4, F5, F41, and F6 (8). Most of these F165-positive E. coli isolates express multiple adhesins with different receptor binding specificities, including recognition of the Gal-Gal or GalNAc-GalNAc moieties and MRHA of human, bovine and/or sheep erythrocytes (9). The expres-

Pathogenic Eschenichia coli strains causing intestinal and extraintestinal infections commonly adhere to eukaryotic epithelial cells as a first step in the colonization of host tissues. Adherence can be mediated by an interaction between specific receptors on the host cells and proteinaceous bacterial appendages called fimbriae (13). Most fimbriae of pathogenic E. coli strains cause mannose-resistant hemagglutination (MRHA) of erythrocytes (34). The various fimbrial adhesins exhibiting MRHA can be distinguished by their receptor binding specificities. In extraintestinal E. coli strains from humans, P, S, M, or X fimbriae -are most commonly found. P fimbriae, found predominantly among uropathogenic E. coli strains, carry the PapG adhesin, which recognizes the a-D-Gal-(1,4)-D-Gal (Gal-Gal) moiety of globoside Qf the P blood group antigen on human erythrocytes and uroepithelial cells (7, 12, 13, 19, 27, 28). These uropathogenic E. coli strains mostly belong to 0 serotypes 01, 04, 06, 016, and 018 (13). Prs fimbriae are closely related to P fimbriae but possess the F or PrsG adhesin, which preferentially binds to the galactose-N-acetyl-a-(1,3)-galactose-Nacetyl (GalNAc-GalNAc) moiety of the Forssman antigen on sheep erythrocytes and in the human kidney (18, 28, 44). Corresponding gene clusters for P and Prs fimbrial adhesins, pap andprs, have been cloned from the chromosome of E. coli J96 (28). Extensive restriction mapping of these clusters has identified structural differences only in the genes encoding the PrsG and PapG proteins, which determine receptor binding specificity (21, 28, 30). Three distinct receptor binding variants of E. coli G adhesins have been defined (30, 43, 44). It has been shown that pap' E. coli clinical isolates associated with pyelonephritis and cystitis exhibit extensive structural differences in the pap-related nucleotide sequences and that some of these isolates harbor multiple * Corresponding author. 2453

2454

INFECT. IMMUN.

MAITI ET AL.

TABLE 1. Adhesin receptor specificities and presence of the DNA sequences related to the pap, sfa and afaI operons in 18 F165-positive E. coli strains used for restriction fragment length polymorphism analysis Receptor-specific

Group

I

II

Porcine intestine

0115

4787a 6389a 1776a 393a

Porcine Porcine Porcine Porcine

0115 0115 0115 0115

2325a

Bovine extraintestine

011

3292b

Bovine intestine Bovine intestine Bovine intestine Bovine intestine Porcine intestine Bovine intestine

015 09 09 09 0101 0?

2313c

Bovine extraintestine

015

2878a 1195a

Bovine intestine Porcine intestine Bovine intestine Porcine intestine Porcine intestine

015 09 09 09 09

3863b 1401a 3984c

215b

1616b 3373b 3719b a

I

c

Serogroup

5131a

7867a

III

Source

Strain

intestine intestine intestine intestine

Genotype

pap sfa

pap afa

MRHA of the following erythrocytes:

Sheep, human group A1P1, and porcine

Bovine and human group A1Pj, 01Pj, and pp

agglutination of: P latex

Forssman latex

-

+

-

+ + + +

-

-

+

+

+ + + pap

Bovine and human group A1Pj, 01Pj, and pp

+ +

+ + + + -

+ +

+ +

Induced septicemia in experimentally inoculated piglets. Not tested in piglets. Did not induce septicemia in experimentally inoculated piglets.

sion of F165 is dependent on the composition of the culture medium (10). In a recent study, we showed that most F165-positive E. coli isolates share similar DNA sequences with the pap operon alone or with thepap operon and either the sfa or the afaI operon (15). Harel et al. (16) cloned a prs-like operon encoding a fimbrial antigen with a binding specificity similar to that of a class III G adhesin (16) from an F165-positive, pap+, sfa+ septicemic E. coli isolate, 4787, and designated it F1651. This fimbrial antigen also agglutinates porcine erythrocytes, unlike the class III G adhesin (16, 44). Another fimbrial antigen, designated F1652, was also purified from the same E. coli isolate, 4787, and found to share amino acid sequence homology with FlC fimbriae, although it differs antigenically from FlC fimbriae (6). We demonstrated that binding specificity for the GalNAc-GalNAc moiety or possession of afa-related or sfa-related DNA sequences is associated with the induction of septicemia in newborn pigs (9). In addition, we showed that an F1651-negative TnphoA mutant of E. coli is less pathogenic in gnotobiotic pigs than its wild-type parent strain (31). The goals of the current study are (i) the structural analysis of the pap-, sfa-, and afaI-related sequences in 18 F165-positive E. coli isolates from diseased calves and piglets; (ii) the determination of the structural variability of the three fimbrial operon-related nucleotide sequences and the correlation between adherence phenotype and structural sequences; and (iii) the comparison of the structural polymorphisms of the fimbrial gene clusters of F165-positive isolates obtained from diseased piglets and calves and containing the pap, sfa, and afaI operons of human uropathogenic E. coli. Our results demonstrate that F1651-positive E. coli isolates of animal origin possess sequences structurally similar to pap- and sfa-related se-

quences, whereas afa-related sequences display structural variability in comparison with the prototype afaI operon. These gene clusters encode adhesins identical or similar to the prototype adhesins and thus presumably play a role in the disease process.

MATERIALS AND METHODS Bacterial strains and plasmids. The 18 F165-positive E. coli isolates used in this study were obtained from the E. coli serotyping laboratory of the Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, Quebec, Canada, and were originally isolated from calves or piglets, either from the intestines of animals for which diarrhea was the most prominent clinical sign or from extraintestinal tissues of animals with septicemia. Eleven of 13 tested isolates induced septicemia in experimentally inoculated piglets (9). All isolates were from different animals and farms. All isolates were aerobactin positive and hemolysin negative. DNA homology with pap, sfa, and afaI operons and adherence to erythrocytes of different animal species of the F165-positive E. coli isolates are listed in Table 1. E. coli J96 and plasmid pPAP5 were kindly provided by B.-I. Marklund (28). Plasmid pANN801-13 was kindly provided by J. Hacker (14). E. coli KS52 and plasmid pIL14 were kindly provided by A. F. Labigne-Roussel (23). Determination of adhesins. E. coli isolates were grown on minimal MD medium at 37°C for the optimal production of F165 (8, 10), and both the hemagglutination and latex bead agglutination assays were performed to determine the presence of various adhesins essentially as described by Fairbrother et al. (8, 10). Hemagglutination of bovine, sheep, porcine, and human erythrocytes was carried out as de-

VOL. 61, 1993

pap-, sfa-, AND afa-RELATED GENES IN E. COLI

scribed previously (11). Bacterial agglutination of P latex particles (Chembiomed Ltd., Edmonton, Alberta, Canada)

PsP

and of human erythrocytes A1Pj and 01Pj was used to determine P adhesin-specific adherence, whereas agglutination of Forssman latex particles (Chembiomed) and of sheep erythrocytes was used to detect Prs adhesin-specific adher-

Li

p1

-IJL-

PAP5

_

I

popC probe

H

lkb

pPAP5

__J

pop EFG probe

I

ence.

S adhesin-specific binding was assayed by use of agglutination of bovine erythrocytes with and without neuraminidase treatment as described by Low et al. (26). In addition, human erythrocytes A1Pj and pp were also treated with neuraminidase and used for agglutination assays. Fresh human erythrocytes A1Pj and pp and bovine erythrocytes were washed twice in phosphate-buffered saline (PBS) (pH 7.4) and once in PBS (pH 5.5), diluted to 3% in PBS (pH 7.4), and treated with neuraminidase (from Vibno cholerae; type III; Sigma Chemical Co., St. Louis, Mo.) at a concentration of 0.2 U for 2 h at 37°C. After treatment, the erythrocytes were washed once in PBS (pH 7.4) and hemagglutination was carried out immediately. As a control, erythrocytes were treated with PBS (pH 7.4) alone before being used in the binding assays. Isolates were considered to express an X adhesin when hemagglutination was positive with human erythrocytes pp. DNA preparation. Total chromosomal DNA was prepared from 5 ml of overnight Luria-Bertani (LB) broth cultures essentially as described by Ausubel et al. (4). Cells were resuspended in 1.7 ml of TE buffer (pH 8.0) (10 mM Tris-HCl, 1 mM EDTA)-100 ,ul of 10% sodium dodecyl sulfate (SDS)-10 ,u of 20-mg/ml proteinase K and incubated for 1 h at 37°C. After incubation, 330 ,ul of 5 M NaCl was added and mixed in, 264 ,1u of CTAB (hexadecyltrimethylammonium bromide)-NaCl solution (10% CTAB in 0.7 M NaCl) was added, and the mixture was incubated at 65°C for 10 min. The mixture was then centrifuged at 14,000 rpm (Sorvall RC5C; Dupont) for 5 min to separate CTAB-protein or CTAB-polysaccharide complexes. The aqueous viscous supernatant was extracted with chloroform-isoamyl alcohol (24:1) and then with phenol-chloroform-isoamyl alcohol. The aqueous viscous supernatant was precipitated with 0.6 volume of isopropanol and washed with 70% ethanol. The resulting pellet was dried and mixed with 300 ,ul of TE buffer. Recombinant plasmid DNAs were purified by CsCl-ethidium bromide density gradient centrifugation (29). Southern blot analysis. The chromosomal DNA was treated with appropriate restriction enzymes, and the DNA restriction fragments were separated by electrophoresis in 0.8 or 0.4% agarose horizontal slab gels and transferred to Zeta probe blotting membranes as recommended by the manufacturer (Bio-Rad Laboratories, Richmond, Calif.) (42). The filters were prehybridized for 4 h at 65°C in a solution containing 5x SSPE (20x SSPE is 3.6 M NaCl plus 0.2 M NaH2PO4 plus 20 mM Na2EDTA [pH 7.7]), 5 x Denhardt's solution (100x Denhardt's solution is 2% Ficoll plus 2% polyvinylpyrrolidone plus 2% bovine serum albumin), and 0.5% SDS. Hybridization with heat-denatured, labelled probe DNA was carried out in fresh hybridization solution overnight at 65°C. Heterologous heat-denatured salmon sperm DNA was added to the hybridization solution to a final concentration of 100 ,ug/ml. The filters were washed in 2x SSC (20x SSC is 3 M NaCl plus 0.3 M Na citrate [pH 7.0]) two times for 5 min each time at room temperature and then in 2x SSC-0.5% SDS two times for 30 min each time at 65°C. Finally, the filters were rinsed in 0.1 x SSC two times for 5 min each time at room temperature. The filters were then exposed to Kodak X-Omat AR film (Eastman Kodak

P|S21

4

1-i L-P..--D aSA M C ECFG

2455

pop llprobe

E

6

P

I I~j ~ D E

C B A

P7

Ev

I F

G S H

Cva

pANN 601-13 (sta)

to 9probe

ota DE probe P2 P3SI

Pt

A

11 L,1,,1

C ata C probe

B

-I I.-4

Pc SI S2

II I

D

plL 14

E

(afal)

I ofa BCDprobe i

i

afa BCDE probe

FIG. 1. Restriction maps of thepap, sfa, and afa operons carried by plasmids pPAP5, pANN801-13, and pIL14, respectively, and the probes used to detect gene clusters related to different regions of the three operons. Restriction endonuclease sites are denoted by vertical lines, and genes are denoted by open boxes. DNA fragments used as probes are denoted by horizontal lines. Restriction endonuclease site abbreviations: B, BamHI; E, EcoRI; Ev, EcoRV; P, PstI; S, SmaI; X, XhoI. Subscripts following restriction enzyme designations refer to consecutive cutting sites.

Co., Rochester, N.Y.) at -70°C. For generation of probes, relevant restriction fragments from appropriate plasmids were isolated from low-melting-temperature agarose (29), purified, and radiolabelled with [a- 2P]dCTP by use of a random priming oligolabelling kit (Pharmacia LKB Biotechnology Inc., Baie d'Urfe, Quebec, Canada) in accordance with the instructions of the manufacturer. DNA probes specific for pap, sfa, and afa and conserved for each of the Pap, Sfa, and Afa adhesin families were derived from the appropriate recombinant plasmids (Fig. 1). These probes were either internal to the operon or spanned the entire operon and were highly specific. For analysis of pap-related sequences, three probes were used: (i) a 0.3-kb highly conserved PstI fragment internal to the papC gene to determine the copy number (Fig. 2); (ii) a 2.25-kb SmaI fragment containing the papEFG genes to detect the presence or absence of a unique BglII site in the adhesin gene, as was found in thepapG gene of strain J96 (Fig. 3); and (iii) an 11-kb EcoRI-BamHI fragment spanning the entire pap operon to determine gene copy number and PstI restriction site polymorphisms (Fig. 4) (2). For analysis of sfa-related sequences, two probes were used: (i) a specific, 0.8-kb PstI fragment containing regions of the sfaD and sfaE genes to determine gene copy number (Fig. SA) and (ii) a 9-kb EcoRV fragment from pANN801-13 and spanning the entire region of the sfa operon to determine gene copy number and internal PstI restriction site polymorphisms (Fig. 5B) (31). In addition, this latter fragment was also used to determine the conserved XhoI and EcoRV restriction sites within the sfa-related sequences. For analysis of afaI-related sequences, three probes from pIL14 containing the afal

2456

MAITI ET AL.

INFECT. IMMUN.

IA 24 3A

1t

26

4A

36

bA

iA

7A 48 5B

6B

,)A

76

XA

11A

1*39B 1UP WE

23.1-

_ 400o

231-

_.9.4--

9.46.6-

9.4-

6.64.4-

4.4-

2.32.0-

2.3-2.0-

23.1g- Sib. 4V

%

4

6.64.42.-

II

III

FIG. 2. Estimation of the copy number of the pap-related sequences in eight representative F165-positive E. coli isolates from groups I, II, and III. DNA was digested with EcoRI (lanes 1A to 11A) or BamHI (lanes 1B to 11B), electrophoresed in 0.4% agarose gels, transferred to nylon membranes, and hybridized with the papC probe. Lanes: 1A, 1B, 4A, 4B, 8A, 8B, E. coli J96; 2A, 2B, 5131 (F+); 3A, 3B, 1776 (F+); SA, 5B, 2325 (P-F-); 6A, 6B, 3292 (F+); 7A, 7B, 1401 (P+F+); 9A, 9B, 1616 (P+); 10A, 10B, 1195 (F+); 11A, 11B, 3373 (P+F+). For size markers, HindIII-cleaved lambda DNA was used. Numbers at left are in kilobases.

operon were used (23): (i) a highly conserved, afa-specific, 0.4-kb PstI fragment internal to the afaC gene to determine the numbers of EcoRI and BamHI DNA fragments (Fig. 6A and B); (ii) a 3-kb SmaI fragment containing the afaED genes and the 3' region of the afaC gene and a 1.1-kb PstI fragment containing part of the afaC gene and the entire afaB gene to determine the conservation of internal SmaI sites; and (iii) a 2.6-kb PstI fragment containing parts of the afaD and afaC genes and a 1.1-kb PstI fragment containing the entire afaB gene and part of the 5' end of the afaC gene to determine PstI restriction site polymorphisms (Fig. 6C).

RESULTS

Phenotypic properties of the F165-positive E. coli isolates. A total of 18 E. coli isolates chosen for this study were originally selected from a group of isolates found to be positive forpap, sfa, and afa probes in colony hybridization (15) (Table 1). These isolates were divided into three groups. Isolates of group I, which were pap' and sfa+ showed MRHA of sheep, pig, and human A1Pj erythrocytes but were MRHA negative for bovine erythrocytes and human 01Pj and pp erythrocytes. In addition, they agglutinated Forssman latex but not P latex beads. Isolates of group II or III, which were pap+ and afa + or only pap', respectively, 1A 2A 34 1B

were all MRHA positive for human erythrocytes (A1P1, 01P1, and pp) and bovine erythrocytes and thus produced one or more X adhesins. In addition, these isolates were MRHA negative for sheep and pig erythrocytes. P latex or Forssman latex agglutination was also found to be variable in both groups II and III (Table 1). Treatment with neuraminidase did not abolish the MRHA of the tested erythrocytes for any isolates from the three groups (Table 1), a result indicating that these isolates may express an adhesin that binds to an unknown receptor moiety different from that bound by the a-sialyl-,-2,3-Gal-specific S adhesin. pap-related gene clusters. (i) Copy number analysis. The structurally related pap and prs operons of E. coli J96, lacking EcoRI and BamHI restriction sites within their functional coding regions, were carried by distinct EcoRI fragments of 15 and 19 kb, respectively, and by distinct BamHI fragments of 26 and 15 kb, respectively (28) (Fig. 2). Thus, for determination of the number of gene clusters related to the entire pap operon, total DNA from each of 18 F165-positive E. coli isolates was digested with EcoRI or BamHI and hybridized with radiolabelled probe papC or papll (Fig. 2). A single copy of pap-related sequences carried either by a single EcoRI fragment (12 to 20 kb) or by a single BamHI fragment (13 to 20 kb) was found in all 13 isolates of groups II and III (Fig. 2). However, in group I

4A 5A 6A 7A 48 5B 68

26 3B

4A 94f 1%A 114 8B 96 lOB 116

-7B 12.2-

g1=

122

12.2:

9!2

611.0--

6.130-

"

1_0

-;

i.0 1.0

I11

III

FIG. 3. Presence or absence of a BglII site in adhesin-related genes in the same eight F165-positive isolates as those shown in Fig. 2. DNA was digested with PstI (lanes 1A to 11A) or PstI-BglII (lanes 1B to 11B), electrophoresed in 0.8% agarose gels, transferred to nylon membranes, and hybridized with the papEFG probe. The arrangements of the strains and their phenotypes are the same as those in Fig. 2. For size markers, a 1-kb DNA ladder was used.

pap-, sfa-, AND afa-RELATED GENES IN E. COLI

VOL. 61, 1993 4A5A 647A 4B

lA 2A 3A 1B 2B 3B

12.29.1

12.2

6-1Z_

.c&

3.0-

0-

lA

586B 7B

2A 3A 4A 18

2R 3B 4R

iC

66

233 wi

9.4 6.6 4.4

2C

4B4--

9

V

-B.

3C

2457 4C

=_

2.3 2.0 I

aan

0.56

2.3

2.0 0.5_ _

0,5_

A

, j

I

Jl

FIG. 4. PstI restriction site polymorphisms in pap-related gene clusters in five isolates from groups I and II. DNA was digested with PstI (lanes 1A to 7A) or PstI-EcoRI (lanes 1B to 7B), electrophoresed, and transferred as described in the legend to Fig. 3 and hybridized with the papll probe. Lanes: 1A, 1B, 4A, 4B, E. coli J96; 2A, 2B, 5131; 3A, 3B, 4787; SA, SB, 7867; 6A, 6B, 3292; 7A, 7B, 1401. For size markers, a 1-kb DNA ladder was used.

isolates, a single BamHI fragment and a single EcoRI fragment were found to hybridize to the papC probe, whereas the papll probe hybridized to two EcoRI fragments and a single BamHI fragment. This result indicates the presence within the pap-related sequences of an internal EcoRI restriction site outside the papC fragment. Probes papC and papli both recognized the same fragment in any one isolate. (ii) Presence or absence of a BglI restriction site in sequences coding for an adhesin, as detected by the papEFG probe. In the control E. coli strain, J96, the papG gene but not the prsG gene contains a BglII site (Fig. 1). In agreement with the literature, the papEFG probe was found to detect distinct PstI fragments of 6.8 and 4.4 kb in strain J96 (Fig. 3, 4

3

2

5

5.03.0O

23.1-

9.4-

4.4-1.0-

2.3

-

2.0-

A

B

FIG. 5. (A) Estimation of the copy number of sfa-related sein group I isolates. DNA was digested with BamHI. The probe was sfaO.8. Lanes: 1, 2, and 3, E. coli 1776, 5131, and 4787, respectively; size markers were HindIll-cleaved lambda DNA. (B) PstI restriction site polymorphisms in sfa-related gene clusters. Lanes: 4, control strain pANN801-13; 5, 6, and 7, E. coli 1776, 5131, and 4787, respectively. For size markers, a 1-kb DNA ladder was used. quences

B

C

FIG. 6. (A and B) Estimation of the copy number of afa-related sequences in group II isolates. DNA was digested with EcoRI (lanes 1A to 4A) or BamHI (lanes 1B to 4B) and hybridized with the afaC probe. Lanes: 1A, 1B, E. coli KS52; 2A, 2B, 215; 3A, 3B, 1401; 4A, 4B, 3863. Size markers were HindIII-cleaved lambda DNA. (C) PstI restriction site polymorphisms in afa-related gene clusters. The probe was afaBCD. The arrangements of the strains are as in panels A and B. For size markers, a 1-kb DNA ladder was used.

lanes 1A, 4A, and 8A) carrying the prsEFG genes and papEFG genes, respectively (2). Double digestion of strain J96 DNA with PstI-BglII and hybridization with the papEFG probe yielded 6.8- and 2.6-kb fragments (Fig. 3, lanes 1B, 4B, and 8B). The 2.6- and 0.9-kb fragments were produced because of the presence of the BglII site in the coding region of papG. To determine whether the BglII site was present in the adhesin-expressing gene of the 18 isolates, we compared their PstI and PstI-BglII restriction profiles with that of J96. Total DNA of each of the 18 F165-positive isolate was digested with PstI and PstI-BglII and hybridized with the papEFG probe. In all five isolates of group I, expressing only the F adhesin, the papEFG probe detected a 4.5-kb PstI fragment and a 4.4-kb PstI-BglII fragment (Fig. 3, lanes 2A, 3A, 2B, and 3B). These results indicate (i) the absence of the Bglll site in the adhesin gene in these isolates and (ii) that the PstI restriction site downstream of the adhesin gene is not conserved, since the band expected for a prsEFG fragment was absent. In contrast, in the isolates of group II, the papEFG probe detected PstI fragments of more variable sizes (Fig. 3, lanes 5A to 7A and SB to 7B). In group II, gene clusters from five isolates yielded a 6.0-kb band, and those from the two other isolates yielded 4.0- and 4.4-kb bands. Upon digestion with PstI-BglII, a 1.9-kb band was observed in all seven isolates of group II. In all six isolates of group III, the papEFG probe detected a 6.0-kb PstI fragment and a 2.0-kb PstI-BglII fragment (Fig. 3, lanes 9A to 11A and 9B to liB). These results indicate that (i) most likely the BglII site is present in the adhesin gene in isolates of groups II and III, as with the papG gene of strain J96; (ii) the presence of the BglII site does not correlate with the expression of the type of adhesin, as isolates agglutinating only F latex also contained a BglII site in the adhesin gene; and (iii) downstream flanking sequences are variable, as detected by nonconserved PstI restriction sites. (iii) PstI restriction site polymorphisms within the gene clusters. Six PstI restriction endonuclease sites are present at similar positions in the pap and prs operons of E. coli J96 (2, 28). Digestion of E. coli J96 total DNA with PstI yielded fragments of 1.7, 1.0, 0.5, 0.3, and 0.1 kb internal to both the pap and the prs operons (Fig. 4, lanes 1A and 4A) (1, 2). Because of the lower limits of resolution of the 0.8% agarose gel and sensitivity of the procedure, the 0.1-kb band could

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not be detected. Southern blots of PstI restriction digests of total DNA of each of the 18 F165-positive isolates and hybridization with the papll probe detected internal bands of 1.7, 1.0, 0.5, and 0.3 kb and junction fragments of variable sizes (Fig. 4). Four isolates of group I produced similarly sized PstI junction fragments of 5.0 and 3.0 kb, whereas one isolate produced only one fragment of 4.5 kb. Five isolates of group II produced 5.0- and 4.4-kb junction fragments, one produced only a 5.0-kb junction fragment, and another produced only a 4.4-kb junction fragment. Five isolates of group III produced 5.0- and 4.0-kb bands, and one produced only a 5.0-kb band. Thus, the six internal PstI restriction sites delineating the 1.7-, 1.0-, 0.5-, and 0.3-kb restriction fragments in the J96 pap and prs operons were conserved in all 18 F165-positive isolates, irrespective of their phenotypes. sfa-related gene clusters. (i) Copy number analysis. There are no BamHI restriction endonuclease sites within the functional limit of the sfa, foc, and sfr fimbrial operons (37, 38). Therefore, for determination of the copy number of sfa-related sequences in the five isolates of group I, total DNA of each of these isolates was cleaved with BamHI and hybridized with the sfaDE probe. The sfaDE probe can also detect foc and sfr determinants. Among the five isolates, four showed a single band ranging from 6.6 to 21 kb (Fig. 5A). This result indicated that flanking sequences of sfarelated gene clusters were not conserved. One isolate (1776), however, showed two bands (Fig. SA, lane 1), of 11 and 6.6 kb, indicating either the presence of a BamHI restriction endonuclease site in the sfaDE region or that this isolate contains two copies of sfa-related sequences. When EcoRVor XhoI-digested DNA of isolate 1776 was hybridized with probe sfaDE, a single band was found to hybridize in each case (data not shown). This result suggests that there is a BamHI restriction site in the sfaDE region rather than that there are two copies of sfa-related sequences in this isolate. (ii) Physical structure, as determined on the basis of PstI restriction site polymorphisms. PstI cleaves the sfa coding region of strain 536 into fragments of 2.6, 1.3, 1.35, 0.7, 0.5, and 2.9 kb (14) but cleaves the sfa-related foc and sfa determinants into seven and five fragments, respectively (37). Thefoc determinant produces seven fragments because of the presence of an additional PstI restriction site in the 2.6-kb fragment at the 3' end (37, 41). In the sfr determinant, however, a larger, 3.4-kb fragment is produced at the 5' end and corresponds to 2.9- and 0.5-kb fragments (38). For determination of whether group I isolates have conserved PstI sites in their gene clusters, total DNA was cleaved with PstI and hybridized with the sfa9 probe, which represents the EcoRV fragment spanning the entire sfa operon. The sizes of the fragments observed are given in Fig. 5B, lanes 4 to 7). Four of the five isolates produced 2.9-, 1.35-, 1.30-, 1.0-, and 0.71-kb bands. One isolate produced 1.35-, 1.30-, and 0.71-kb bands in addition to 2.0- and 1.0-kb bands. This result demonstrates that the PstI restriction sites producing 1.35-, 1.3-, and 0.71-kb fragments are conserved in the same positions as in the three prototype operons. In addition, the presence of the 2.9-kb fragment in four isolates suggests the conservation of the two restriction sites also present in the sfa and foc determinants. (iii) Relationship withfoc. Both the sfa and thefoc operons are flanked by two EcoRV sites which, upon digestion with EcoRV, produce an 8-kb fragment in both cases. The foc operon contains in its adhesion-encoding region an XhoI site that is absent from the sfa operon. The EcoRV-digested DNA of isolates of group II, when probed with the sfa9

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probe, hybridized with an 8-kb band. The XhoI-digested DNA, however, when probed with the sfa9 probe, hybridized with a 16- or a 19-kb band (data not shown). Double digestion with EcoRV-XhoI produced a 7.4-kb band in all isolates, suggesting that the 8-kb EcoRV fragment possessed an XhoI site, as in the foc operon (36, 37). afaI-related gene clusters. (i) Copy number analysis. Genetic determinants of afimbrial adhesin Afal share considerable structural homology with those of three other adhesins, Dr, F1845, and AfaIll (19, 22). An internal EcoRI site has not been found within the functional limits of the operons of each of these adhesion systems (22, 45). When total DNA of each of the group II isolates was cleaved with the EcoRI restriction endonuclease and probed with the afaC probe, all the isolates showed a 4.9-kb band, in contrast to the 16-kb band present in afaI-positive strain KS52 (Fig. 6A, lanes 1A to 4A). BamHI digestion and hybridization with the afaC probe, on the other hand, resulted in bands of variable sizes (Fig. 6B, lanes 1B to 4B). These results indicate that (i) afaI-related sequences differ internally from the prototype operons and (ii) flanking sequences of these gene clusters are heterogeneous. (ii) Physical structure, as determined on the basis of internal PstI and SmaI restriction site polymorphisms. Total DNA of each of the isolates of group II was digested with PstI and probed with the afaBCD probe (Fig. 1). None of the isolates exhibited PstI fragments with the same electrophoretic mobility as those identified in the AfaI (2.6, 1.1, and 0.4 kb), F1845 (0.54 and 0.43 kb) (41), and Dr (2.28 kb) operons (22, 33, 45). Instead, the isolates displayed PstI fragments of variable sizes (Fig. 6C, lanes 2C to 4C). Of the seven isolates, three produced a 4.0-kb band, two produced 4.0and 2.6-kb bands, one produced 4.07- and 1.1-kb bands, and one produced 6.2- and 3.0-kb bands. Only one internal SmaI site is present in the afaI operon (22). When SmaI- and PstI-digested DNA of group II isolates was hybridized with the afaBCDE probe (Fig. 1), fragments with different electrophoretic mobilities were found (data not shown). These hybridization experiments showed that (i) the internal sequences in the afaI-related gene clusters are very heterogeneous and (ii) the bordering sequences both downstream and upstream from the afaIrelated sequences are variable, since a difference in the sizes of the PstI fragments was found with the afaBCD and afaBCDE probes. In addition, results from the afaBCDE probe hybridization experiment also suggested heterogeneity in bordering sequences, as demonstrated by the differences in the SmaI fragments. DISCUSSION We describe the structural polymorphisms of the pap-, sfa-, and afaI-related gene clusters and the correlation of these polymorphisms with adherence phenotype properties in 18 F165-positive E. coli isolates from cattle and swine. Physical characteristics of pap-related gene clusters in relation to adhesin-mediated agglutination. A single copy of the pap-related gene cluster in each of our 18 F165-positive isolates may be responsible for the expression of each of the three phenotypes (P+, F+, and P+F+). Two isolates each of groups II and III, each possessing a single copy of the pap-related gene cluster, agglutinated both P latex and Forssman latex beads. The expression of either the P adhesin or the F adhesin is normally associated with a single gene copy (2), and the simultaneous expression of both the P and the F adhesins has been associated with the presence of

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at least two copies in some uropathogenic E. coli strains causing pyelonephritis, such as strain J96. This strain expresses both a class I G adhesin (binds to globotriaosylceramide [GbO3] and demonstrates the P+ phenotype) and a class III G adhesin (binds to GbO5 and demonstrates the F+ phenotype), encoded by two separate gene clusters,pap and prs, respectively. However, some strains isolated from cases of human urosepsis display both the P and the F adhesin phenotypes and contain only a single copy of thepap operon (2). The P latex and Forssman latex agglutination observed in our four isolates may thus indicate that the singlepap- or prs-like gene cluster in each isolate encodes an adhesin recognizing both P and F structures (24, 25). Similarly, it was demonstrated that a single pap copy in strains AD110 (F72) and IA2 (Fll) encodes an adhesin that can bind to globotetrasylceramide, Forssman glycolipid, heptaglucosylceramide (globo-A), and Gal-Gal-latex in vitro and that agglutinates sheep erythrocytes (18). Isolates of both groups II and III agglutinated bovine erythrocytes, and the majority of these isolates were from diseased calves. Thus, these isolates may carry a G adhesin(s) different from that carried by human isolates, which do not agglutinate bovine erythrocytes. The G adhesins of bovine isolates may have adapted to recognize cells of this species. Two isolates of group II (pap+ and afa+) agglutinated neither P latex nor Forssman latex beads. These two isolates may possess pap-related DNA sequences expressing adhesins with receptor binding moieties slightly different from Gal-Gal or GalNAc-GalNAc. As observed for the J96 pap and prs operons (2, 3), no PstI restriction site polymorphisms were found internally in thepap-related gene clusters in our isolates. The conservation of internal PstI sites in pap-related gene clusters in clinical isolates causing pyelonephritis and cystitis in humans has been shown (1, 3). However, some pap' strains isolated from human urinary tract infections and expressing neither the P nor the F adhesin lacked the internal 1.0-kb PstI fragment and instead produced a novel 0.8-kb band. This result was probably due to a small deletion in the papC orpapD gene that may have abolished the expression of the adhesins (2, 3, 32). In contrast, we did not observe any fragment with a different electrophoretic mobility in our two P-F- isolates. Isolates expressing the F adhesin lacked a BglII site that was also absent in the prsEFG region of the prs operon (2, 16). However, the presence of a conserved BgllI site in the papG-related sequences of group II and III isolates expressing both P and Forssman latex agglutination phenotypes indicates that there is no correlation between the expression of these latter adhesins and the absence of a BglII site in the adhesin genes. The lack of conservation of the DNA sequences adjacent to the pap-related gene clusters in the same arrangement as was found in strain J96 suggests that a variation involving the DNA sequences flanking the pap-related sequences is frequent. It has been hypothesized that pap-related sequences may be found on transposons and be transferred directly to different sites on the same chromosome or to plasmid or phage vectors and then to the chromosomes of different isolates (40). A Southern blot analysis of plasmids from human uropathogenic and animal septicemic isolates, however, failed to demonstrate pap-related operons on plasmids (16, 20). It is possible that sequences flanking thepap-related sequences undergo rearrangements through inter- or intrachromosomal recombination, thus generating a diversity of flanking sequences. Physical structures of sfa-related gene clusters in group I

isolates in relation to adhesin-mediated agglutination. Although three related operons, sfa, foc, and sfr, share extensive genetic homology, minor differences exist among them in the regions that code for the major fimbrial subunit (14, 38, 41). The five sfa+ isolates of group I, which induced septicemia in piglets (9), did not agglutinate bovine erythrocytes, unlike E. coli 536, which expresses the S adhesin. Copy number analysis with the sfaDE probe detected a single copy of the sfa-related sequences in isolates of group I. In contrast, Blum et al. (5) and Ott et al. (35) found multiple copies of foc determinants in E. coli 06 extraintestinal isolates from humans. Differences in the fragment sizes of isolates of group I suggested that flanking sequences of the sfa-related gene clusters are not conserved and that the positions of the gene clusters are highly variable. Structural analysis with restriction enzymes PstI, EcoRV, and XhoI suggested that group I septicemic isolates contain sequences that are more closely related to foc than to sfa or sfr. This suggestion confirms recent evidence that at least one of these isolates produces fimbrial component F1652, which closely resembles the product of foc, FlC (6). Physical structures of afa-related sequences in group II isolates in relation to adhesin-mediated agglutination. The use of the afaC probe demonstrated that, unlike the results obtained with the afaI operon, a single 4.9-kb EcoRI band hybridized in all seven group II isolates (Fig. 6A). In addition, hybridization of this probe to differently sized BamHI fragments (Fig. 6B) suggested that afa-related sequences contain internal EcoRI sites conserved in similar positions among' the isolates. Hybridization of probe afaBCD with PstI-digested chromosomal DNA from the seven isolates detected an unexpectedly large number of PstI polymorphisms within the internal and flanking sequences (22). Hybridization of probe afaBCDE with SmaIdigested DNA showed that in none of the seven isolates were the types of bands expected for afaI found (data not shown). These structural analyses suggested that the internal afa-related sequences as well as the flanking sequences in our isolates are different from the sequence's in the AfaI, AfaII, F1845, and Dr systems and that the position or the orientation of the genes in afa-related sequences may be different from that in the prototype operon from KS52 (22, 23). In conclusion, we have demonstrated that F165-positive E. coli isolates from diseased animals possess gene clu'sters very similar to thepap and foc sequences of human extraintestinal E. coli isolates, whereas sequences related to afa are very different. Sequences structurally related to pap or prs of human E. coli isolates have not been described for bovine and porcine E. coli isolates. The observation that a single copy of the pap gene expressing a single adhesin can recognize more than one receptor with different affinities suggests a strategy used by the pathogen to adhere to different host tissues expressing different receptors, or isoreceptors' (30). In addition, group I isolates possessing both pap- and sfa-related sequences are phenotypically and genotypically- homogeneous and thus may be clonally related. The occurrence of pap-, sfa-, and afa-related sequences in E. coli from diseased animals suggests that these sequences may be transmitted from animal isolates to human isolates or vice versa via the horizontal transfer of genetic material and subsequent recombination (30). It has also been suggested that a genetic potential for pathogenicity has arisen among a small number of unrelated clones. Differences in the 0 serotypes of F165-positive animal isolates and human uropathogenic or septicemic isolates further strengthen the

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above-described hypothesis. If transmission of the sequences occurs for E. coli isolates among animal species, then selection pressure in a new environment may result in the evolution of a completely or partially changed adhesin(s). ACKNOWLEDGMENTS This work was supported by grants to J.H. and J.M.F. from the Conseil de Recherches en Sciences Naturelles et an Genie du Canada (OGP0025120 and OGP002294, respectively). S. Maiti was a recipient of a predoctoral studentship from Ministere de l'Enseignement Sup6rieur et de la Science du Gouvernment du Quebec. REFERENCES 1. Arthur, M., R. D. Arbeit, C. Kim, P. Beltran, H. Crowe, S. Steinbach, C. Campanelli, R. A. Wilson, R. K. Selander, and R. Goldstein. 1990. Restriction fragment length polymorphisms among uropathogenic Escherichia coli isolates: pap-related sequences compared with rm operons. Infect. Immun. 58:471479. 2. Arthur, M., C. Campanelli, R. D. Arbeit, C. Kim, S. Steinbach, C. E. Johnson, R. H. Rubin, and R. Goldstein. 1989. Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherchia coli. Infect. Immun. 57: 314-321. 3. Arthur, M., C. E. Johnson, R. H. Rubin, R. D. Arbeit, C. Campanelli, C. Kim, S. Steinbach, M. Agarwal, R. Wilkinson, and R. Goldstein. 1989. Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli. Infect. Immun. 57:303-313. 4. Ausubel, M. F., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology, vol. 1. Wiley Interscience, New York. 5. Blum, G., M. Ott, A. Gross, and J. Hacker. 1991. Virulence determinants of Escherichia coli 06 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques. Microb. Pathog. 10:127-136. 6. Dubreuil, J. D., and J. M. Fairbrother. 1992. Biochemical and serological characterization of Escherichia coli fimbrial antigen F1652. FEMS Microbiol. Lett. 95:219-224. 7. Ekback, G., S. Morner, B. Lund, and S. Normark. 1986. Correlation of genes in the pap gene cluster to expression of globoside-specific adhesin by uropathogenic Escherichia coli. FEMS Microbiol. Lett. 34:355-360. 8. Fairbrother, J. M., A. Broes, M. Jacques, and S. Lanviere. 1989. Pathogenicity of Escherichia coli 0115:KV165" strains isolated from pigs with diarrhea. Am. J. Vet. Res. 50:1029-1036. 9. Fairbrother, J. M., J. Harel, C. Forget, C. Desautels, and J. Moore. 1993. Receptor binding specificity and pathogenicity of Escherichia ccli F165-positive strains isolated from piglets and calves and possessingpap-related sequences. Can. J. Vet. Res. 57:53-55. 10. Fairbrother, J. M., R. Lallier, L. Leblanc, M. Jacques, and S. Larivinre. 1988. Production and purification of Escherichia coli fimbrial antigen F165. FEMS Microbiol. Lett. 56:247-252. 11. Fairbrother, J. M., S. Lariviere, and R. Lallier. 1986. New fimbrial antigen F165 from Eschenichia coli serogroup 0115 strains isolated from piglets with diarrhea. Infect. Immun.

51:10-15. 12. Garcia, E., A. M. Hamers, H. E. N. Bergmans, B. A. M. Van Der Zeist, and W. Gaastra. 1988. Adhesion of canine and human uropathogenic Escherichia coli and Proteus mirabilis strains to canine and human epithelial cells. Curr. Microbiol. 17:333-337. 13. Hacker, J. 1990. Genetic determinants coding for fimbriae and adhesins of extraintestinal Eschenchia coli. Curr. Top. Microbiol. Immunol. 151:1-27. 14. Hacker, J., G. Schmidt, C. Hughes, S. Knapp, M. Marget, and W. Goebel. 1985. Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic 06:K15:H31 Eschenichia coli strain. Infect. Immun. 47:434 440. 15. Harel, J., F. Daigle, S. Maiti, C. Desautels, A. Labigne, and

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J. M. Fairbrother. 1991. Occurrence of pap-, sfa-, and afarelated sequences among F165-positive Escherichia coli from diseased animals. FEMS Microbiol. Lett. 82:177-182. 16. Harel, J., C. Forget, J. Saint-Amand, F. Daigle, D. Dubreuil, M. Jacques, and J. Fairbrother. 1992. Molecular cloning of a determinant coding for fimbrial antigen F1651, a Prs-like fimbrial antigen from porcine septicaemic Eschenchia coli. J. Gen. Microbiol. 138:1495-1502. 17. Hull, S., S. Clegg, C. Svanborg Eden, and R. Hull. 1985. Multiple forms of genes in pyelonephritogenic Escherichia coli encoding adhesins binding globoseries glycolipid receptors. Infect. Immun. 47:80-83. 18. Johanson, I., R. Lindstedt, and C. Svanborg. 1992. Roles of the pap- andprs-encoded adhesins in Eschenichia coli adherence to human uroepithelial cells. Infect. Immun. 60:3416-3422. 19. Johnson, J. R. 1991. Virulence factors in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128. 20. Johnson, J. R., S. L. Moseley, P. L. 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. 21. Karr, J. F., B. J. Nowicki, L. D. Truong, R. A. Hull, J. J. Moulds, and S. I. Hull. 1990. pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney. Infect. Immun. 58:40554062. 22. Labigne-Roussel, A. F., and S. Falkow. 1988. Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates. Infect. Immun. 56:640-648. 23. Labigne-Roussel, A. F., D. Lark, G. Schoolnik, and S. Falkow. 1984. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect. Immun. 46:251-259. 24. Lindstedt, R., N. Baker, P. Falk, R. Hull, S. Hull, J. Karr, H. Leffler, C. Svanborg Eden, and G. Larson. 1989. Binding specificities of wild-type and cloned Escherichia coli strains that recognize globo-A. Infect. Immun. 57:3389-3394. 25. Lindstedt, R., G. Larson, P. Falk, U. Jodal, H. Leller, and C. Svanborg. 1991. The receptor repertoire defines the host range for attaching Eschenchia coli strains that recognize globo-A. Infect. Immun. 59:1086-1092. 26. Low, D. A., B. A. Braaten, G. V. Ling, D. L. Johnson, and A. L. Ruby. 1988. Isolation and comparison of Escherichia coli strains from canine and human patients with urinary tract infections. Infect. Immun. 56:2601-2609. 27. Lund, B., F. Lindberg, B.-I. Marklund, and S. Normark. 1987. The papG protein is the alpha-D-galactopyranosyl-(1-*4)-betaD-galactopyranose-binding adhesin of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 84:5898-5902. 28. Lund, B., B.-I. Marklund, N. Stromberg, F. Lindberg, K.-A. Karlsson, and S. Normark. 1988. Uropathogenic Escherichia coli can express serologically identical pili of different receptor binding specificities. Mol. Microbiol. 2:255-263. 29. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 30. Marklund, B.-I., J. M. Tennent, E. Garcia, A. Hamers, M. Baga, F. Lindberg, W. Gaastra, and S. Normarlk 1992. Horizontal gene transfer of the Escherichia coli pap andprs pili operons as a mechanism for the development of tissue-specific adhesive properties. Mol. Microbiol. 6:2225-2242. 31. Ngeleka, M., M. Jacques, B. Martineau-Doize, J. Harel, and J. M. Fairbrother. 1993. Pathogenicity of an Eschenichia coli 0115:K"V165' mutant negative for F1651 fimbriae in septicemia of gnotobiotic pigs. Infect. Immun. 61:836-843. 32. Norgren, M., S. Normark, D. Lark, P. O'Hanley, G. Schoolnik, S. Falkow, C. Svanborg-Ed6n, M. Baga, and B. E. Uhlin. 1984. Mutations in E. coli cistrons affecting adhesin to human cells do not abolish Pap pili fiber formation. EMBO J. 3:1159-1165. 33. Nowicki, B., J. P. Barrish, T. Korhonen, R. A. Hull, and S. I. Hull. 1987. Molecular cloning of the Eschenichia coli 075X

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adhesin. Infect. Immun. 55:3168-3173. 34. Orskov, F., and I. Orskov. 1983. Serology of Escherichia coli fimbriae. Prog. Allergy 33:80-105. 35. Ott, M., L. Bender, G. Blum, M. Schmittroth, M. Achtman, H. Tschape, and J. Hacker. 1991. Virulence patterns and long-range genetic mapping of extraintestinal Escherichia coli Kl, K5, and K100 isolates: use of pulsed-field gel electrophoresis. Infect. Immun. 59:2664-2672. 36. Ott, M., J. Hacker, T. Schmoll, T. Jarchau, T. K. Korhonen, and W. Goebell. 1986. Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections. Infect. Immun. 54:646-653. 37. Ott, M., M. Hoschutzky, K. Jann, I. Van Die, and J. Hacker. 1988. Gene clusters for S fimbrial adhesin (sfa) and FlC fimbriae (foc) of Escherichia coli: comparative aspects of structure and function. J. Bacteriol. 170:3983-3990. 38. Pawelzik, M., J. Heesemann, J. Hacker, and W. Opferkuch. 1988. Cloning and characterization of a new type of fimbria (S/FlC-related fimbria) expressed by an Escherichia coli 075: K1:H7 blood culture isolate. Infect. Immun. 56:2918-2924. 39. Plos, K., T. Carter, S. Hull, R. Hull, and C. Svanborg Eden. 1990. Frequency and organization of pap homologous DNA in relation to clinical origin of uropathogenic Escherichia coli. J. Infect. Dis. 161:518-524.

40. Plos, K., S. I. Hull, R. A. Hull, B. R. Levin, I. 0rskov, F. 0rskov, and C. Svanborg-EdEn. 1989. Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer. Infect. Immun. 57:1604-1611. 41. Riegman, N., R. Kusters, H. V. Veggel, H. Bergmans, P. V. B. E. Henegouwe, J. Hacker, and I. Van Die. 1990. FlC fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits. J. Bacteriol. 172:1114-1120. 42. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 43. Stromberg, N., B.-I. Marklund, B. Lund, D. liver, A. Hamers, W. Gaastra, K.-A. Karisson, and S. Normark. 1990. Hostspecificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal-alphal-4Gal-containing receptors. EMBO J. 9:2001-2010. 44. Stromberg, N., P. Nyholm, I. Pascher, and S. Normark 1991. Saccharide orientation at the cell surface affects glycolipid receptor function. Proc. Natl. Acad. Sci. USA 88:9340-9344. 45. Swanson, T. N., S. S. Bilge, B. Nowicki, and S. L. Moseley. 1991. Molecular structure of the Dr adhesin: nucleotide sequence and mapping of receptor-binding domain by use of fusion constructs. Infect. Immun. 59:261-268.

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