INFECTION AND IMMUNITY, Apr. 1983, p. 265-2720019-9567/83/040265-08$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 40, No. 1

Contribution of Adhesion to,Bacterial Persistence in theMouse Urinary Tract

L. HAGBERG,1 R. HULL,2 S. HULL,2 S. FALKOW,3 R. FRETER,4 AND C. SVANBORG EDANI*Department of Clinical Immunology, University of Goteborg, Goteborg, Sweden'; Department ofMicrobiology and Immunology, Baylor School of Medicine, Houston, Texas 770302; Department of

Microbiology, Stanford School of Medicine, Stanford, California 943053; and Department of Microbiology,University of Michigan, Ann Arbor, Michigan 481094

Received 9 November 1982/Accepted 8 December 1982

The affinity of uropathogenic Escherichia coli to kidneys and bladders ofexperimentally infected mice was shown to be determined in part by the adhesiveproperties of the infecting bacteria. Mice were infected with various pairwisecombinations of two homogeneic sets of bacteria: (i) mutants derived from ahuman pyelonephritis E. coli isolate which were selected to express either or bothadhesins specific for globoseries glycolipid receptors or for "mannosides"; and(ii) transformants of a normal fecal isolate which harbored recombinant plasmidsencoding the genes for one or the other adhesin or which harbored only the vectorplasmid. The relative efficiency of survival of the strains to be compared wasevaluated in each animal by plating on selective media of samples of homogenizedkidneys and bladders taken 24 h after intravesical inoculation. The presence ofadhesins specific for globoseries glycolipid receptors, which mediate the in vitromannose-resistant attachment to human and mouse uroepithelial cells, enhancedbacterial recovery from both kidneys and bladders of infected animals. Theaddition to the infecting strain of adhesins binding mannoside residues furtherimproved bacterial recovery from the bladder, but not from the kidney. Themutants and transformants with adhesins binding only mannosides were recov-ered in higher numbers from the bladder than those expressing adhesins specificfor the globoseries glycolipids only. There was apparent selection in vivodecreasing expression of mannoside binding adhesins in the kidneys, but not inthe bladders, of animals infected with the mutant expressing both types ofadhesins. Regardless of adhesive properties, the mutants of the pyelonephritisisolate were recovered in significantly higher numbers than the fecal isolate withadhesins encoded on recombinant plasmids. We conclude that the adhesiveproperties in part determine the localization and retention of bacteria in the mouseurinary tract. However, the addition of adhesins to a commensal E. coli strain wasnot sufficient to confer colonization capacity comparable to that of a pyelonephri-tis strain.

Adherence to specific components of the mu-cosal surfaces is considered a prerequisite formicrobial colonization (3, 7) or infection (8, 23)or both. Adhesive capacity has been shown tobe necessary although not sufficient for Esche-richia coli carrying the K88 adhesin to inducediarrhea in piglets (23).

E. coli that cause acute pyelonephritis in theunobstructed human urinary tract possess multi-ple virulence factors (26). The capacity of E. colibacteria to attach to human uroepithelial cells invitro is related to the severity of infection in-duced in the patient (8, 24, 26; H. Lomberg,L. A. Hanson, B. Jacobsson, U. Jodal, H.Leffler, and C. Svanborg Eden, submitted forpublication). The attachment of the majority ofpyelonephritogenic E. coli (11, 13) as well as

their capacity to agglutinate human erythrocytesmay be explained by specific binding to globo-series glycolipid receptors (GGR) in the mem-branes of these cells (11, 13, 14). Most uropatho-genic E. coli also bind to mannose-containing,but as yet unidentified, receptors that are pre-sent, e.g., in urinary slime (20). This binding ismeasured in vitro as mannose-reversible aggluti-nation of guinea pig erythrocytes.

Pili (fimbriae) can be the bacterial surfacestructures, adhesins, binding both to GGR (25a)and to mannose-containing receptors (21). Thegenes encoding these pili of uropathogenic E.coli so far studied have been identified as dis-tinct loci on the bacterial chromosome (9, 27).The genes, pil, that encode pili binding to "man-nosides" as well as those genes, pap, that gov-

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TABLE 1. Properties of the mutants and transformantsAdhesion to uroepithelial cells (mean no.

Hemagglutination to cells bacteria/cell)E. coli strain Guinea pig Mouse Other properties

Human Na- Coat- Human Rattive eda CBA BALB/c

Mutants (M)GR12, parent GSb MS GS 98 NTc 64 NT All mutantspap pil+ were 075Knt,d

HU734 mutant GS MS GS 65 48 61 55 serum resist-Mpap+pil+ ant, hemoly-

HU742 mutant MS 0 37 11 6 sin nega-Mpap-pil+ tive, ColV

HU824 mutant GS GS 67 0 40 37 positiveMpap+pili

Transformants (T)J96, donor GS MS GS 81 NT 42 NT 04K6, hemoly-pap+pil+ sin positive

506, recipient 0 0 0 0 016K1, hemoly-Tpap-pil- sin negative

506, pACYC 184 0 0 0 0 016K1, hemoly-Tpap-pil- sin negative

506, pRHU845 GS GS 59 8 38 45 016K1, hemoly-Tpap+pilU sin negative

506, pSH2 MS MS MS >100 >100 >100 >100 016K1, hemoly-Tpap-pil+ sin negative

a Coated with globotetraosylceramide.b GS, Globotetraosylceramide-sensitive agglutination.c NT, Not tested.d Knt, K antigen not typable.

ern biosynthesis of pili which specifically bind toGGR have both been isolated by recombinantDNA methods (10). In addition, a series ofhomogeneic mutants of a wild-type pyelonephri-togenic E. coli deficient in pil or pap or neithergene has been obtained (25).

In this study we report the use of definedgenetic strains of E. coli to examine the patho-genesis of urinary tract infection in a mousemodel. The data show that both adhesion mech-anisms may play a significant role in infection,but probably in different regions of the urinarytract.

MATERIALS AND METHODSAnimals. Female CBA mice were purchased from

Anticimex, Stockholm, Sweden, and BALB/c micewere obtained from W. Murphy, Department of Mi-crobiology and Immunology, University of Michigan,Ann Arbor. The animals were housed five each to acage, fed ad libitum, and used for infection at 6 to 8weeks of age.

Bacteria. The E. coli strains used in the study arelisted in Table 1. Although the presence of nonpiliformadhesins on the strains cannot be completely ruledout, the designations pap' for GGR-binding adhesinsand pil+ for mannoside-binding adhesin will beused.

(i) Mutants. HU734, HU742, and HU824 are mu-tants of a wild-type E. coli strain, GR12, isolated froma patient with acute pyelonephritis. HU734, a lac-mutant of GR12, was isolated after treatment withnitrous acid (17). HU742 and HU824 were derivedfrom HU734 after treatment with N-methyl-N-nitro-N-nitrosoguanidine (2). The mutants (M) retained fromthe GR12 parent all of the phenotypic traits availablefor testing: they had identical electrophoretic mobilityof 15 chromosomally encoded enzymes (22); threeplasmids, the largest identified as ColV; the sameserotype (075Knt) (15) and biotype; and were resist-ant to the bactericidal effect of serum (19). HU734retained the two parental pili types, pap and pil, andwas designated Mpap+ pil'. HU824 bound only toGGR and was assumed to be pap' pil- (Mpap pil-),whereas HU742 only exhibited mannose-reversiblebinding and was assumed to be pap-pil' (Mpap-pil+). Derivates of these strains resistant to streptomy-cin or nalidixic acid were selected by serial passage ongradient plates. E. coli HU734 Strr and HU824 Strrwere resistant to 1 mg of streptomycin per ml; HU824Nalr and HU742 Nalr were resistant to 50 ,ug ofnalidixic acid per ml. The resistant mutants wereconsidered stable when they grew in similar numberson media with or without antibiotic after one passageon antibiotic-free medium. The possible risk that se-lection of antibiotic resistance had altered other bacte-rial properties, e.g., those important for in vivo infec-tivity, was tested by mixed infections with the originalstrains and the resistant mutants.

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ADHESION IN THE MOUSE URINARY TRACT 267

(ii) Transformants. The cloning of the pil (for man-noside-binding adhesin) and pap (for GGR-bindingadhesin) genes, resulting in expression of pili andadhesive properties, was originally performed in an E.coli K-12 recipient strain from strain J% (10). E. coliK-12 was not suitable for the present studies because itwas quickly eliminated from the mouse urinary tract.For this reason E. coli 506, which had been isolatedfrom the stools of a healthy child and was phenotypi-cally Pil-Pap-, was transformed with three plasmids:the vector pACYC184 (4); pRHU845, carrying the papgene(s) necessary for expression of mannose-resistant(MR) pili inserted into the chloramphenicol resistancegene of pACYC184; and pSH2, carrying the pil genefor mannose-sensitive (MS) pill inserted into the tetra-cycline resistance gene of pACYC184. Transform-ants (T) were obtained (4) encoding either pap506(pRHh845), designated Tpap+pilU, or pil506(pSH2), designated Tpap-pil+. The plasmidpRHU845 contained a 6.7-megadalton chromosomalrestriction fragment, contained the pap genes insertedinto the EcoRI site, and inactivated the chlorampheni-col resistance gene of the vector pACYC184 (10). Theplasmid SH2 contained an 8-megadalton chromosomalDNA insert including the pil genes SalI site inactivat-ing the tetracycline resistance gene of pACYC184. Thestrain 506P(pACYC184), designated Tpap-pil-, con-tained the plasmid pACYC184 without insertions andretained resistance to chloramphenicol and tetracy-cline. To allow detection out of a mixture a mutant ofTpap-pil-, resistance to 50 g±g of nalidixic acid per mlwas selected. The transformants acquired the proper-ties of attachment and hemagglutination as expectedbased upon the plasmid each had received.

Infection procedure. Various pairwise combinationsof the strains were used to inoculate the urinarybladders of female mice. Mice were chosen becauseattachment of pyelonephritogenic E. coli strains tomouse uroepithelial cells in vitro was similar (Table 1).The transmission of bacteria to the urinary tract fromthe intestine has not been reconstructed in animalmodels. Direct injection of bacteria into the bladder,without obstructive manipulations, was considered tobe the least artificial way to induce experimentalinfection in mice. Mixed inocula have been used by anumber of authors (6, 16) to correct for variation in theresponse of individual animals, when comparing thevirulence of bacterial strains. Mixed inocula wereprepared in phosphate-buffered saline with strainsdiffering in adhesins and distinguishable by resistanceto different antibacterial agents. The recovery fromkidneys and bladders was quantitated by plate countsof the bacteria in homogenized tissue sampled 24 hafter infection. The relative recovery of each inoculumcomponent from each animal was taken as a measureof the role of its adhesin in initiating the colonization ofparticular tissue.The precise details concerning the infection model

are provided in the accompanying paper (8). In brief,the animals were anesthetized by ether inhalation. Thebladder was emptied by gentle compression of theabdomen. Immediately thereafter, a soft polyethylenecatheter (outer diameter, 0.61 mm; Kebo Grave, Swe-den) adapted to a 0.4- by 20-mm gauge needle on a 1-ml tuberculin syringe (Asik, Denmark) was transureth-rally inserted into the bladder. A 0.05-ml amount of theappropriate microbial inoculum was injected. The

catheter was withdrawn immediately, and no furthermanipulations were performed. The ether anesthesialasted for about 30 s, after which time the animals wereallowed food and drink ad libitum.

Inoculum. Bacterial strains used to inoculate theanimals were maintained on appropriate antibioticagar plates. For infection studies, they were passagedonce on antibiotic-free tryptic soy agar (TSA), exceptMpap+pil+ and Mpap-pil+, which were grown onstatic modified Luria broth (10 g of tryptone, 5 g ofyeast extract, 10 g of sodium chloride per 1,000 ml ofdistilled water, and 2.5 ml of 10%o CaC12) to expressMS adhesins. After harvest the bacterial cell densitywas adjusted on the basis of a standard curve ofabsorbance at 597 nm to a concentration of 1010bacteria per ml by dilution in phosphate-buffered sa-line (300 mosmol/liter, pH 7.2).

Before mixing the binding properties of the inocu-lum components were tested. Agglutination of humanand guinea pig erythrocytes with or without a-methylmannoside (final concentration, 10 mg/ml) was per-formed as described previously (9), and designated asMS if reversed by a-methyl mannoside or MR ifunaffected by a-methyl mannoside. Specific binding togloboseries glycolipids was tested by using the recep-tor-coated erythrocyte technique (13) and designatedglobotetraosylceramide sensitive. The capacity of theinoculum strains to attach to mouse uroepithelial cellswas tested as follows: uroepithelial cells from thesediment of urine pooled from 30 to 50 mice werewashed once and suspended in phosphate-bufferedsaline. About 105 cells were mixed with 100 ijl of thebacterial suspension in a total volume of 1 ml. Afterincubation and washing as previously described (24),adhesion was determined as the mean number ofbacteria attached to 40 epithelial cells.The inoculum strains were then mixed in equal

parts. The exact concentration of each strain wasdetermined by viable counts on TSA and TSA contain-ing the antibiotics to which the inoculum componentswere resistant. For the mutants HU734 Strr, HU824Nal' or Strr, and HU742 Nalr the number of coloniesexpected on TSA was the sum of those found withstreptomycin and with nalidixic acid present. The 506PNair strain grew equally well on TSA-nalidixic acid,TSA-chloramphenicol and TSA-tetracycline, since itcontained the cloning vector. The transformants of E.coli 506 Tpap+pil- and Tpap-pil+ were resistant totetracycline and chloramphenicol, respectively. Forthe transformants the number of colonies on the TSA-nalidixic acid plates was subtracted from those withtetracycline and chloramphenicol to quantitateTpap+pil- and Tpap-pil+, respectively, out of a mix-ture with Tpap-pil- Nalr.

Bacterial recovery from tissues. The animals were

sacrificed by cervical dislocation 24 h after infection.Kidneys and bladders were removed aseptically andhomogenized in a Teflon tissue grinder (A. ThomasCo., Philadelphia, Pa.). Viable counts of 0.05 ml ofserial dilutions of kidney and bladder homogenateswere performed on TSA and TSA containing therelevant antibiotics. After adjustment for dilution therecovery of each strain was calculated as R = (concen-tration in the tissue)/(concentration in the inoculum).The relative recovery of strains a and b in the inocu-lum mixture was compared by calculation of therecovery ratio, RaIRb, in each animal.

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TABLE 2. Comparison of in vivo infectivity of parent strains and antibiotic-resistant mutants

Inoculum mixture Kidney Bladder

Strain a Strain b Nimalf R.lRba 95% Cjb pc No. of RIRb 95% Cl P

Mpap+pil+ Mpap+pil+ 11 1.66 0.62-4.89 NSd 15 0.87 0.32-2.40 NSStrs Strr

Mpap-pil+ Mpap-pil+ 9 5.75 2.29-22.8 <0.05 12 5.50 1.10-25.3 <0.05Strs Strr

Mpap-pil+ Mpap-pil+ 9 1.82 0.17-15.5 NS 13 1.12 0.28-4.57 NSNals Nalr

Mpap+pil- Mpap+pil- 12 2.00 0.48-12.0 NS 14 0.78 0.34-1.78 NSStrs Strr

Mpap+pil- Mpap+pil- 12 1.62 0.78-3.23 NS 13 2.39 0.51-11.22 NSNals Nalr

a The recovery (R) of each inoculum component (a and b) was estimated by viable counts on TSA withappropriate antibiotics and with no antibiotic of homogenized tissue and calculated as a percentage of theconcentration of each strain in the inoculum. The geometric mean of the recovery ratios of the two strains isshown.

b CI, Confidence interval.c P, Level of significance determined by Student t test.d NS, Not significant.

Statistical evaluation. Due to large positive skewingof the recovery values a transformation to logarithmicscale was undertaken, and the geometric mean was

used for statistical analyses. Differences in ability topersist in the mouse urinary tract were evaluated ingroups of mice by the Student t test of the recoveryratios.

RESULTS

Bacterial binding properties in vitro. The hem-agglutinating and adhesive properties of thestrains and transformants are shown in Table 1.Mpap+pil+, Mpap+pil-, and Tpap+pil- all at-tached to mouse uroepithelial cells as shown inTable 1 and agglutinated human erythrocytes inan MR manner. The adhesins were specific forGGR, as shown by their ability to induce MRagglutination of guinea pig erythrocytes after,but not before, coating with globotetraosylcera-mide. Mpap+pil+, Mpap-pil+, and Tpap-pil+all produced MS agglutination of guinea pigerythrocytes. Tpap-pil+, in addition, agglutinat-ed human erythrocytes in an MS manner. Mpap-

-pil+ attached poorly, and Tpap-pil+ attachedstrongly, to mouse uroepithelial cells. The adhe-sion of Mpap-pil+ and Tpap-pil+ was reversedby a-methyl mannoside. Parenthetically, we no-ticed that Mpap+pil+, Mpap-pil+, and Tpap-pil+ were bound to strands of urinary mucus,

but this association could not be quantitated. Nodifference in attachment to uroepithelial cellsfrom female CBA and BALB/c mice was seen.The in vitro binding to a variety of target cells ofthe mutants and transformants have been report-ed elsewhere (C. Svanborg Eddn, R. Hull, S.Falkow, and H. Leffler, Nutr. Res., in press).

Control of the antibiotic-resistant mutants. Theantibiotic-resistant mutants were compared withtheir parents for ability to persist in the mouseurinary tract (Table 2). With mixed inocula, onlyMpap-pil+ Strr showed impaired survival com-

pared with the parent, and it was not usedfurther. The in vitro binding of the resistantmutants showed the same level and specificityas the parents (data not shown).

TABLE 3. Relative recovery from kidneys and bladders of E. coli mutantsa

Inoculum mixture Kidneys BladdersMousestrain No. of Rlb 9%C o fRlb 9%CStrain a Strain b animals R/Rb 9 C animalsof

Mpap+pil+ Mpap+pilU CBA 17 0.51 0.18-1.48 NS 19 3.09 1.66-5.75 <0.01Strr Nalr BALB/c 14 2.29 0.80-4.02 NS 15 4.07 2.29-7.24 <0.001

Mpap+pil+ Mpap-pil+ CBA 11 10.5 2.13-51.3 <0.05 18 3.63 1.74-7.76 <0.01Strr Nair BALB/c 23 53.7 15.1-170 <0.001 23 34.7 10.7-112 <0.001

Mpap-pil+ Mpap+pil- CBA 15 0.16 0.059-0.43 <0.01 19 5.75 2.24-14.8 <0.01Nalr Strr BALB/c 11 0.56 0.32-0.77 <0.01 17 3.16 1.95-6.46 <0.01

a Mpap+pil+, Mpap-pil+, and Mpap+pil- carried adhesins specific for both GGR and mannoside receptors,for only mannoside receptors, and for only GGR, respectively. Experimental conditions and abbreviations are asin Table 2.

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ADHESION IN THE MOUSE URINARY TRACT 269

TABLE 4. Relative recovery of wild-type and transformant E. coli strains, from kidneys and bladders ofCBA and BALB/c micea

Inoculum mixture Kidneys Bladders

Strain a Strain b strain No. of RRb 95% Cl | No. | RlRb 95% Cl Panimals ~~~~~~~~~~~~~~~~~~~~~~animalsRIb 9%C

Wild-type Tpap- CBA 18 2.19 0.25-10.9 NS 21 4.07 2.08-7.94 <0.01strain 506 pil-

Tpap-pil' Tpap- BALB/c 10 2.50 0.95-5.88 NS 20 111.11 29.4-384 <0.001pil- CBA 18 0.63 0.15-4.35 NS 18 4.35 1.99-9.55 <0.01

Tpap+pil- Tpap- BALB/c 11 4.35 1.13-16.7 <0.05 20 4.25 1.72-11.2 <0.01pil- CBA 19 3.33 1.27-9.09 <0.05 19 2.63 0.85-9.09 NS

Tpap-pil+ Tpap+ BALB/c 11 0.76 0.4-1.44 NS 17 29.51 9.77-89.1 <0.001I pil CBA 4 10 4.36 1.51-12.6 <0.05

a Experimental conditions and abbreviations are as in Table 2.b Insufficient number of animals.

Role of adherence for bacterial persistance inkidneys and bladders. (i) Mutants. The results ofmixed infections with Mpap+pil+ Strr andMpap+pil- Nalr with Mpap+pil+ Strr andMpap-pil+ Nalr and of Mpap+pil- Strr withMpap-pil+ Nalr are shown in Table 3. Data forCBA and BALB/c mice are shown in parallel(see below), although the experiments, exceptfor Mpap+pil- Strr and Mpap-pil+ Nalr, weredone on different days. In the kidneys, no differ-ence in bacterial recovery was found betweenMpap+pil+ Strr and Mpap+pil- Nalr, whereasMpap-pil+ Nalr was recovered in lower num-bers. In the bladder Mpap+pil+ Strr persistedbetter than either Mpap+pil- Nalr or Mpap-pil+Nalr. Mpap-pil+ Nalr was recovered significant-ly more from the bladder than was Mpap+pil-Strr. The results are consistent with the interpre-tation that the pap genes encoding the adhesin(s)specific for GGR provided a significant advan-tage for bacterial persistance in the mouse kid-ney. The pil genes did not offer enhanced orimpaired bacterial persistance in the kidney, butincreased the recovery from the urinary bladder.Mpap-pil+ was recovered in numbers abouttwo- to sixfold lower than Mpap+pil- from thekidneys. In the urinary bladder Mpap+pil+ hadabout a three- to fourfold advantage overMpap-pil+.

(ui) Transformants. The recovery of E. coli 506wild type and transformants from kidneys andbladders of CBA and BALB/c mice is shown inTable 4. Tpap-pil-, containing only the cloningvector, had impaired infectivity compared withthe wild-type strain, possibly due to adverseeffects of the plasmid. Tpap+pil- persisted bet-ter in the kidney than Tpap-pil-, and Tpap+pil-and especially Tpap-pil+ persisted in the blad-der more efficiently than did Tpap-pil-.

Difference in susceptibility between BALB/cand CBA mice. Variations in glycolipid composi-tion of kidney tissue (1) prompted a comparisonbetween CBA and BALB/c mice (Fig. 1). The

same inoculum mixture of E. coli Mpap+pil-Strr and Mpap-pil+ Nalr was used to infectgroups of 10 mice of either strain. Mpap+pil-Strr was found to dominate moreover Mpap-pil+ Nalr in CBA as compared withBALB/c mouse kidneys (P < 0.05) (Fig. la). Nodifference in recovery from the urinary bladdersof the two mouse strains was seen (Fig. lb). Thecumulative sample distribution illustrates theshift in ratio of recovery of Mpap-pil+ Nalr/Mpap+pil- Strr. Mpap+pil- Strr dom-inated in the kidney, whereas Mpap-pil+ Nalrdominated in the bacterial population recoveredfrom the bladder.

Binding properties of the bacteria recoveredafter infection. The hemagglutinating propertiesof the bacteria recovered from kidneys andbladders were tested directly from the agarplates as well as after overnight cultures in Luriabroth. The results for Mpap+pil+ are shown inTable 5. Before infection, the strain expressedpili specific for GGR and mannosides. Mostcolonies recovered from the kidney expressedonly the GGR adhesins, whereas the bladderisolates from the same animals expressed bothtypes. After subculture for 2 weeks in the labo-ratory, all strains isolated from the animals againexpressed both adhesins.

Role of carrier strain properties. The averagerecovery of the mutants of the wild-type strainGR12 from kidneys and bladders was 0.003% ofthe inoculum, whereas the recovery of the trans-formants of the normal fecal strain 506 was0.00012%. To directly quantitate the contribu-tion of bacterial host cell properties other thanadhesion, the mutant strains and transformantsexpressing pili with the same receptor specificitywere compared in mixed inocula (Table 6). Inkidneys and bladders of all animals the mutantderivatives of the wild-type strains persisted inhigher numbers than the transformants express-ing the same adhesins. The difference in recov-ery between mutants and transformants with the

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270 HAGBERG ET AL.

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FIG. 1. Susceptibility of CBA (0) and BALB/c (*mice to infection with a mixture of Mpap+pil- Strr aneMpap-pil+ Nalr. The logs of the ratios of the tw(strains are shown in a cumulative sample distribution(a) The advantage for Mpap+pil- Stri over Mpap-pil'was higher in CBA than in BALB/c mouse kidneys, aseen by the lower recovery ratio. (b) There was nc

difference between the mouse strains in recovery oMpap+pil- Stri and Mpap-pil+ Nalr from mouslbladders.

same adhesin was larger than between homogenic strains differing only in adhesins.

DISCUSSIONIt is usually assumed that E. coli causing

human urinary tract infection originate from theintestine and reach the urinary tract by theascending route. Adhesive properties have beensuggested to select bacteria capable of reachingand colonizing the normal urinary tract and tcinfluence the level of infection (24, 26). Themajor types of adhesins identified on uropathogenic E. coli may be differentiated by the effectof a-methyl mannoside. The binding of bacteriato GGR, which obviously is not inhibited b)mannose, accounts for the attachment of mosi

strains causing kidney infection (11, 14). Binding

to mannosides occurs for most uropathogenicstrains regardless of the level of infection, mostoften on strains from cystitis patients (9). Bothtypes of adhesins are commonly coexpressed byE. coli isolates from pyelonephritis after in vitroculture.For this investigation genetic technology has

been used to construct homogenic strains of E.coli differing in their adherence properties. Therole of each adhesin type for the persistence ofbacteria in the kidney and urinary bladder was

quantitatively assessed in an in vivo mousemodel. An E. coli strain (GR12) isolated from a

patient with acute pyelonephritis was used as

the parent from which mutants were recoveredafter chemical mutagenesis. HU734, a lac- mu-

tant of GR12 recovered after treatment withnitrous acid, retained both types of parentaladhesins. After treatment of this strain withnitrosoguanidine two mutants were selected,one that lacked the adhesins specific for GGR(Mpap-pil+) and another that lacked the MSadhesin (Mpap+pil-). The precise nature of themutations which resulted in the loss of expres-sion of either adhesin has not been identified.Secondary mutations resulting in deficientexpression or binding properties of the adhesinswere unlikely because the mutants retained thespecificity and intensity of in vitro adherenceand agglutination of the parent (Table 1). How-ever, changes in other bacterial properties can-not be excluded. Traits associated with viru-lence in human urinary tract infection, such as

d serotype and the resistance to the bactericidal° effect of serum, were unaltered. The strains+ harbored the ColV plasmid. The bacterial strainss of the second homogenic set were derivatives of0 a nonadherent fecal isolate that had been trans-

formed with recombinant plasmids constructedby inserting segments of DNA encoding eitherthe MR or MS adhesin from a pyelonephritisisolate into the vector plasmid pACYC184. The

TABLE 5. Hemagglutination pattern and recoveryof E. coli Mpap+pil+a

Hemagglutination pattern No. infected/total'

Before infection After sacrificeKid- Blad-

Human Guinea Human Guinea ney derpig pig

GSb MS GS 13/18 4/30GS MS 4/18 26/30

1/18 0/30a The results are the means of two experiments with

30 animals infected in each group with each strain.b GS, Globotetraosylceramide-sensitive agglutina-

tion.c Number of organs from which hemagglutinating E.

coli were recovered/total number of infected organs.

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ADHESION IN THE MOUSE URINARY TRACT 271

TABLE 6. Relative recovery from kidneys and bladders of mutants and transformants with the same adhesinspecificity, but differing in other virulence factorsa

Inoculum mixture Kidneys BladdersMouse

Strana Stran bstrain No. of No. ofStrain a Strain b animals RaRb 95% CI P animals RaRb 95% CI P

Mpap- Tpap- CBA 10 85.1 22.9-295 <0.001 18 70.8 27.5-182 <0.001pil+ pil+

Mpap+ Tpap+ CBA 6 12.5 1.23-128 0.05 10 5.0 2.12-8.91 <0.01pil- pil-a Experimental conditions and abbreviations are as in Table 2.

exact product of the pil and pap genes withadhesin function has not been identified.

In comparing the relative recoveries from thekidneys, strains with the GGR adhesins enjoyedan advantage over strains with the MS adhesinalone or with neither adhesin type. In four offivecomparisons, this advantage was statisticallysignificant. The MS adhesins, in contrast,seemed to give little advantage in the kidney.The presence of MS adhesins in a strain neitherincreased nor decreased the recovery from kid-ney tissue, compared with the nonadheringtransformants. These results strongly favor theconclusion that the attachment to GGRs is asignificant factor in the ability of bacteria topersist in the mouse urinary tract. These conclu-sions are in agreement with results previouslyreported (25) where the recovery of Mpap+pil-(E. coli HU824) was decreased after preincuba-tion of bacteria with globotetraose.The importance of adhesins binding to the

globoseries glycolipids for bacterial persistencein the kidney was further illustrated by two otherfindings of this investigation. Bacteria tested forhemagglutination properties immediately uponrecovery from samples of homogenized tissueindicated suppression of MS adhesins, whereasbladder isolates from the same animal expressedboth GGR and MS adhesins. The mechanismbehind this selection might be retention of bacte-ria in bladder mucus, a phase variation inducedin the kidney tissue or a selective phagocyteclearance of bacteria with MS adhesins. Supportfor a role of GGR adhesins was also provided bythe higher relative recovery from kidneys ofMpap+pil- over Mpap-pil+ in CBA mice com-pared with that in BALB/c mice. No differencewas seen in in vitro adherence to bladder cells orin colonization of the bladder. The larger recov-ery of the GGR-binding mutant from the kidneyof CBA mice may relate to the composition ofkidney glycolipids (1). The content of neutralceramide-containing glycolipids was 15 mg/100mg of kidney tissue for CBA compared with 6mg/100 mg for BALB/c mice. The proportion ofceramide-tritrexoside and ceramide-N-acetyl-hexosaminyl-tritrexoside was about 80% in CBA

mouse kidney. Kidney tissue from BALB/c micecontained about 40o ceramide tritrexoside andonly trace amounts of the aminoglycolipid (1).This suggests a lower amount of glycolipidscontaining the proposed recognition site sharedby globoseries glycolipids, the Galal-+4Galpdisaccharide, in BALB/c mice than in CBAmice. A difference in receptor density linked todifferent susceptibility to infection has also beenproposed in humans (Lomberg et al., submittedfor publication). The globoseries glycolipids areantigens in the P blood group system (5). Eryth-rocytes from individuals of the P1 blood groupcontain higher amounts of glycolipids sharingthe Gala1- 4GalP disaccharide than do erythro-cytes from individuals of the P2 blood groupphenotype (Lomberg et al., submitted for publi-cation). The P1 blood group is overrepresentedamong girls with recurrent pyelonephritis with-out reflux. This increased susceptibility has notyet been shown to be due to higher receptordensity in kidneys or urinary tract epithelium ofthese patients.

In contrast, bacterial survival in the mouseurinary bladder was enhanced by both adhesintypes. The mutants or transformants with MSadhesin were recovered at significantly greaterfrequencies than were the homogenic strainsexpressing only the GGR adhesin. The largestrelative recovery from the bladder in any experi-ment was seen with Tpap-pil+ compared withTpap-pil-. Of all strains tested, Mpap+pil+with both adhesins was recovered from thebladder most efficiently.The greater differential recovery of mutants

than of transformants expressing the same adhe-sins (Table 6) indicates a requirement for addi-tional virulence factors to establish infection inthe urinary tract. The transformants, both de-rived from the rough strain K-12 and the smoothfecal strain 506, adhered in vitro as well as, orbetter than, the mutants derived from a pyelone-phritis isolate, yet the transformants were at asignificant disadvantage in vivo.The use of mixed inocula to directly compare

the role of single virulence factors for pathogen-esis was also employed by Smith and Lingood

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272 HAGBERG ET AL.

(23) in a study that is somewhat analogous to thepresent one. In contrast to that work, the pres-ent study was carried out with nonconjugativeplasmid vectors and is therefore not subject tothe criticism that in vivo plasmid transfer mighthave obscured the interpretation. Smith andLingood found that both K88 adhesins and en-

terotoxin were needed for disease. Uropatho-genic E. coli differ from enterotoxigenic E. coliin that no "second-step" virulence factor com-parable to the enterotoxin has been defined. The"pathogenic personality" of E. coli strains caus-

ing acute pyelonephritis can be distinguished bycertain serotypes (26), in which adhesive proper-ties, serum resistance, hemolysin production(18), and possibly as yet undefined virulencefactors coexist. The urinary tract mouse modeldescribed here may provide a tool for investigat-ing the roles of these suspected virulence factorsin the development of pyelonephritis.

ACKNOWLEDGMENTSThis study was supported by grants from the Swedish

Medical Research Council (no. 215), The Medical Faculty,University of Goteborg, The Ellen, Walter and Lennart Hes-selman Foundation for Scientific Research, and The Volks-wagen Foundation and by National Science Foundation grantPCM 8015722 and Public Health Service grant Al 10885-07from the National Institutes of Health. S.H. was a traineeunder Public Health Service grant 07149-02 from the NationalInstitutes of Health, and R.H. was a fellow of the NationalScience Foundation and the National Institutes of Health.The excellent technical assistance of Inga Engberg and the

typing aid of A.-C. Malmefeldt are greatly appreciated.

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