Vol. 26, No. 10JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1988, p. 2087-20910095-1137/88/102087-05$02.00/0Copyright © 1988, American Society for Microbiology

Immunoblot Analysis of Serologic Response to Outer MembraneProteins of Escherichia coli in Elderly Individuals with

Urinary Tract InfectionsL. E. NICOLLE,'* E. UJACK,2 J. BRUNKA,' AND L. E. BRYAN2'3

Division ofInfectious Diseases3 and Departments of Medicine and Microbiology and Infectious Diseases,2University of Calgary, Calgary, Alberta T2N 1N4, and Department of Medical Microbiology,

University of Manitoba, Winnipeg, Manitoba R3E OW3,' Canada

Received 23 December 1987/Accepted 14 July 1988

We used immunoblotting to examine the serologic antibody responses to outer membrane proteins (OMP) ofEscherichia coli in both symptomatic and asymptomatic elderly subjects with urinary tract infections. Controlswith no present or past urinary tract infections showed variable weak immunoglobulin G (IgG) antibodies toOMP of infecting strains. Elderly individuals with asymptomatic infections demonstrated antibody to bothlipopolysaccharide (LPS) and OMP of their infecting strain, with consistent cross-reactivity to OMP of otherinfecting strains. Young females with acute pyelonephritis showed an IgG response to LPS and OMP withcross-reactivity to OMP of other strains. Elderly individuals with symptomatic invasive infections had strongreactions to both LPS and OMP in specimens collected during the acute phase, generally with an increase inintensity in specimens from convalescent patients. They also demonstrated extensive cross-reactivity to LPSand OMP from all other infecting strains. IgM antibody was not observed in any patients. These data confirmother reports of low levels of antibodies to OMP of E. coli in normal populations. Asymptomatic bacteriuriain this population is associated with antibody responses to the LPS and OMP of the infecting strain. Elderlyindividuals with invasive infections had initial reactions to the infecting strain with an apparent increase inintensity during convalescence. Antibodies to the major OMP appear to be broadly cross-reactive.

Urinary tract infection occurs frequently in elderly indi-viduals (7), with the prevalence approaching 50% for resi-dents in extended care institutions. Escherichia coli is themost common infecting organism in elderly women andsecond after Proteus mirabilis in elderly men (1, 10, 11). Inyounger individuals, invasive infection with E. coli is asso-ciated with development of specific antibodies to the lipo-polysaccharide (LPS) and outer membrane proteins (OMP)of the infecting strain (4, 6). Initial responses are immuno-globulin M (IgM) and subsequent IgG. Individuals withasymptomatic infection may have some antibodies to LPSand OMP, but these are generally of low titer. The humoralimmune system of the elderly has certain features whichdiffer from that of a younger population (16). These includea decreased response to primary antigenic challenge, anincrease in autoantibodies, and a relatively normal responseto a secondary antigenic challenge. The humoral immuno-logic response of elderly individuals to E. coli urinary tractinfection has not been described.Fever is a common manifestation of infection in institu-

tionalized elderly persons (12), many of whom are bacteriu-ric, and it may be diagnostically impossible to exclude agenitourinary source. Since invasive urinary infection in theelderly should lead to a humoral antibody response, anti-body measurement could be an objective test for invasiveinfection. With the antigenic heterogeneity of LPS and therelative conservation of OMP antigens among Enterobac-teriaceae (5, 13), we postulated that the OMP of E. coli couldserve as an antigen in a diagnostic test for invasive infection.As an initial approach, we undertook a preliminary charac-terization by immunoblotting of the serologic immune re-sponse to OMP in elderly individuals with symptomatic or

* Corresponding author.

asymptomatic infections with E. coli and report our obser-vations here.

MATERIALS AND METHODS

Clinical specimens. Clinical specimens were obtained fromhospitalized patients. The four elderly noninfected controlsincluded three males 75, 80, and 82 years old and one female84 years old. These patients were hospitalized for orthopedicprocedures (2), small bowel perforation, and investigation ofprostatic carcinoma and had no prior history of urinary tractinfection or genitourinary intervention; they had negativeurine cultures when serum specimens were obtained. Inaddition, they had not received antimicrobial therapy withinthe prior 6 months. Three females 16, 25, and 36 years oldhospitalized with a clinical illness consistent with acutepyelonephritis, including fever, flank pain, and a urineculture positive for E. coli, were included as comparativelyyoung symptomatic patients, since the immunologic re-sponse in this group has been at least partially characterized(4, 6). Initial specimens were obtained when the diagnosis ofacute pyelonephritis was made, and convalescent-phasespecimens were obtained 7, 9, and 11 days thereafter. Theasymptomatic bacteriuric elderly patients included four fe-males 68, 85 (n = 2), and 98 years old. Three of these werewomen resident in an extended care facility who had per-sistent asymptomatic bacteriuria with E. coli of over 1 yearduration, including one woman infected with two distinctserotypes. The fourth asymptomatic subject was an inpatienthospitalized for investigation of back pain with asympto-matic E. coli bacteriuria from whom specimens were ob-tained initially and 3 weeks later. The duration of bacteriuriain this individual was unknown. All subjects with asympto-matic bacteriuria were bacteriuric when serum specimenswere obtained. Four elderly symptomatic patients with

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invasive urinary tract infections included two females 68 and81 years old and two males 75 and 90 years old. Hospitaladmission was for increasing confusion (2), hip fracture, andmyocardial infarct. Three patients had bacteremic hospital-acquired urinary tract infections, and the fourth had postcys-toscopy sepsis. Acute-phase specimens were obtained atdiagnosis, and convalescent-phase serum samples were ob-tained 7, 21, 23, and 28 days later.

Species identification of organisms was done with the API20E system (API Laboratory Products Ltd., St. Laurent,Quebec, Canada). None of the patients studied had chronicindwelling catheters. Serum specimens were stored at -70°Cuntil immunoblotting was performed.

Preparation of OMP. Organisms isolated from urine werestored in glycerin at -70°C and subcultured on agar oncebefore being used for OMP preparation. Outer membranepreparations were obtained by the method of Hancock andNikaido (3). Briefly, an overnight culture of 20 ml of theinfecting organism was grown at 37°C, diluted to 2 liters infresh medium (Tryptic soy broth), and grown with vigorousshaking at 37°C for 4 to 6 h. Four liters of cells was harvestedby centrifugation at 4°C, and all subsequent operations werealso performed at 4°C. The cells were washed with 30 mMTris hydrochloride (pH 8.0; Tris buffer; Hoechst, La Jolla,Calif.) and suspended in 10 ml of 20% (wt/vol) sucrose in 50mM Tris buffer plus 0.2 mM dithiothreitol (Sigma ChemicalCo., St. Louis, Mo.). The cells were passed twice through aFrench press at 15,000 lb/in2, 2 ml of hen egg white lysozyme(1 mg/ml) was added, and the solution was kept for 10 min inan ice bath in a cold room and shaken twice. A proteaseinhibitor, phenylmethylsulfonyl fluoride (Sigma) was addedat 0.174 mg/ml of cells. The mixture was sonicated in ice atpower 7 three times for 20 s each time with a model 350 celldisruptor (Branson Sonic Power Co., Danbury, Conn.) witha 1/8-in. (1 in. = 2.54 cm) tapered microtip on a 1/2-in. horn.Cell debris was removed by centrifugation at 3,500 rpm for10 min (1,464 x g; RC2-B; Ivan Sorvall, Inc., Norwalk,Conn.), and the supernatant was decanted into a sucrosestep gradient containing 1 ml of 70% (wt/vol) sucrose, 2.5 mlof64% (wt/vol) sucrose, 2.5 ml 58% (wt/vol) sucrose, and 2.5ml 52% (wt/vol) sucrose in Tris buffer. The tubes were thencentrifuged at 45,000 rpm for 14 h (1,993 x g; Sorvall RC2-BGSA rotor). Bands observed between the 70 and 64% layerswere removed by dropwise collection from the bottom, andindividual samples were diluted in 10 ml of 50 mM Tris plus0.2 mM dithiothreitol and centrifuged at 45,000 rpm for 1 h.This wash was repeated. The resultant pellets were sus-pended in 50 mM Tris buffer plus 0.2 mM dithiothreitol andfrozen at -70°C. The amount of protein present was mea-sured by the method of Lowry et al. (9).

Immunoblotting. Protein samples were thawed, and pairedspecimens, each containing 50 ,ug of protein, were prepared.Proteinase K (GIBCO Canada, Burlington, Ontario) wasadded at 50 ,ug/10 FLI to alternate samples to enhancedetection of LPS and confirm the protein nature of specificbands, and the samples were incubated for 60 min in a 60°Cwater bath. All samples were then boiled for 3 min in a waterbath. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis was performed by the method of Laemmli (8).Electrophoresis of OMP was done at 50 mA of constantcurrent with Tris-glycine (pH 9.3) plus 0.1% sodium dodecylsulfate buffer for approximately 4 to 5 h. OMP were thentransferred electrophoretically to a nitrocellulose sheet (Bio-Rad Laboratories, Richmond, Calif.) by the method ofTowbin et al. (15). A Bio-Rad Trans-Blot Cell apparatus(Bio-Rad, Mississauga, Ontario, Canada) was used with

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FIG. 1. Immunoblot of sera from three noninfected controls(lanes 2 to 4) with OMP preparations for one strain of E. coli. Thefirst of each pair of lanes was prepared without proteinase Kdigestion, and the second was prepared with proteinase K. Thestained OMP are shown in blot 1, and blot 5 is the control withoutserum from a patient.

blotting performed at 30 V with 9 A of constant currentovernight at 4°C (pH 8.3) in Tris-glycine buffer with 20%(vol/vol) methanol. After protein transfer, the nitrocellulosesheet was immersed in 1.2% fish gelatin (Northland Prod-ucts) in Tris-saline for 1.5 h at 37°C with slow shaking withserum diluted 1/100 with Tris-saline-fish gelatin buffer. Afterthis incubation, the sheets were washed five times in 1.2%fish gelatin in Tris-saline buffer and then incubated with 2%fish gelatin with peroxidase-labeled goat anti-human IgG orIgM (Mandel Scientific Co., Edmonton, Alberta, Canada)and rinsed five times with Tris-saline buffer. Color wasdeveloped by immersion for 20 min in a solution of 3 mg of4-chloro-1-naphthol (Bio-Rad, Mississauga, Ontario, Can-ada) per ml in methanol with 5 volumes of Tris-saline bufferand an H202 concentration of 3%. Molecular weight stan-dards were run with each gel and stained on the nitrocellu-lose sheet to document protein transfer. In addition, poly-acrylamide gels were stained after immunoblotting toconfirm transfer of proteins from the gel. Nonspecific reac-tions due to adherence of the conjugate to the nitrocellulosewere excluded by controls run without serum samples frompatients.

RESULTS

Elderly controls. Sera obtained from elderly noninfectedcontrols were immunoblotted with the outer membranepreparations of bacteria isolated from eight elderly subjects,four with symptomatic infections and four with asympto-matic infections. Figure 1 shows the reactions of sera fromthree noninfected subjects with one OMP preparation. Mostcontrols showed weak reactions with major OMP (molecularweight, 30,000 to 45,000) from most isolates. In addition,reactions with the LPS contaminating the protein prepara-tion were apparent for some controls.Young females with pyelonephritis. Immunoblotting with

both acute- and convalescent-phase sera from young femaleswith acute pyelonephritis demonstrated antibodies to bothOMP and LPS from the infecting strains. Figure 2 showsresults with OMP preparations from two organisms andserum from one patient. When the patient's serum was

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A 1 2a b a b

B 1 2a b a b

C 1 2 D i1 2a b a b a b a b

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FIG. 2. Immunoblotting of acute-phase (panel B) and convalescent-phase (panel C) serum specimens from a young woman with acutepyelonephritis against OMP from the infecting E. coli strain (lanes 2) and E. coli strains isolated from a symptomatic subject (lanes 1). PanelA shows the OMP, and panel D shows the control without serum. For each pair of lanes, lane a was prepared without proteinase K and laneb was prepared with proteinase K digestion.

blotted with OMP preparations from other E. coli strains,cross-reactions to major OMP in the 36-kilodalton region, aswell as some other OMP, were apparent in all cases. Inaddition, occasional cross-reactions with contaminating LPSwere present. Differences in the intensities of reactions withOMP (Fig. 2) between acute- and convalescent-phase serawere apparent for some, but not all, patients. No reactionswith IgM antibodies were observed.Asymptomatic elderly subjects. Elderly subjects with

asymptomatic bacteriuria had antibodies to the OMP, andoccasionally the LPS, of the infecting strains (Fig. 3, lanes a

and b, respectively). All asymptomatic individuals alsodemonstrated cross-reactivity with OMP of organisms iso-lated from other subjects, both symptomatic and asympto-matic, with a variable degree of reactivity to LPS contami-nating the preparation. No differences in blotting reactionswere observed between two specimens obtained from oneasymptomatic patient (data not shown). No bands wereidentified when goat anti-human IgM antibodies were tested.Symptomatic elderly subjects. Elderly individuals with

invasive urinary tract infections showed initial strong immu-noblotting reactions to both the OMP and LPS of the

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FIG. 3. Immunoblotting of sera from two women who had asymptomatic bacteriuria with E. coli for over 1 year against OMP preparationsfrom four E. coli strains. For each pair of lanes, lane a was prepared without proteinase K digestion and b was prepared with proteinase K.Lanes 2, organism isolated from the patient whose serum was tested in panel B; lanes 3, organism isolated from the patient whose serum was

tested in panel C. Panel A shows OMP from the four organisms, and panel D is a control without test serum.

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FIG. 4. Immunoblotting with acute-phase (a) and convalescent-phase (b) sera from one elderly male with a bacteremic urinary tractinfection with his own (A) and another (B) infecting E. coli strain.Lanes 1 were prepared with proteinase K digestion, and lanes 2were prepared without proteinase K. Lane S contained molecularsize markers. Kd, Kilodaltons.

organisms with which they were infected in both acute- andconvalescent-phase sera. For three of the four subjects,there was a consistent apparent increase in the intensities ofthe reactions between acute- and convalescent-phase sera(Fig. 4). For one individual with no convalescent-phaseincrease in intensity, the convalescent-phase serum samplewas obtained only 7 days after the bacteremic episode.When the serum was cross-reacted with organisms fromother subjects, three demonstrated strong cross-reactionswith OMP, with apparently stronger intensity in convales-cent-phase specimens. Cross-reacting antibodies to the con-taminating LPS in the preparation were more consistentlyseen with sera front symptomatic patients than in sera fromasymptomatic patients.

DISCUSSION

Elderly persons with bacteriuria, particularly those ininstitutions, tend to have persistent or recurrent infections(10, 11), and thus these individuals have had prior exposureto uropathogens. In the small number of elderly individualsstudied here, antibodies to both LPS and major OMP wereidentified in a noninfected control group with no knownhistory of urinary infection, although these reactions wereweak. This observation is similar to reports on other agegroups (14) and is postulated to reflect antigenic stimulationby normal gut flora.

Elderly individuals with asymptomatic bacteriuria ap-peared to have a more intense antibody response to OMPthan did noninfected individuals. In addition, antibody to theOMP of the infecting organism was cross-reactive with theOMP of other uropathogenic E. coli strains. The elderlyindividuals with invasive genitourinary infections studiedshowed initial intense immunoblotting reactions to both theOMP and contaminating LPS of the infecting organisms andthe heterologous organisms. Convalescent-phase sera gener-

ally demonstrated an apparent increase in the intensity of theIgG response to the infecting organism. There was widecross-reactivity with the OMP of other E. coli strains iso-lated from urinary tract infections. Whereas the quantitationof differences in antibody responses on the basis of theseimmunoblots must be interpreted with caution because ofthe variability of conditions between blots, the consistencyof the differences observed suggests that the observationsare valid.Shand et al. (14) used immunoblotting to examine the

antibody responses to OMP in two older individuals withindwelling catheters and asymptomatic bacteriuria with P.mirabilis and Klebsiella pneumoniae. Their observationswith respect to the major OMP were similar to thosereported here, although acute- and convalescent-phase serawere not tested. In addition, they also demonstrated cross-reactivity of antibodies to the major OMP among Enterobac-teriaceae species as we did among E. coli strains. Theseobservations are consistent with data which show substan-tial similarity in the antigenicity of major OMP among theEnterobacteriaceae (5, 13).Our purpose was to examine the antibody response to

OMP in elderly patients with urinary tract infections. How-ever, since some LPS contamination remained in the OMPpreparations, some observations about immunoblotting re-actions with LPS were possible. Whereas antibodies to theLPS of infecting strains in both symptomatic and asympto-matic infections were consistently observed, extensivecross-reaction among the LPS of different strains in elderlyindividuals with symptomatic infections also occurred. Thisdiffered from observations on young women with acutepyelonephritis, in whom little cross-reaction with LPS wasapparent, although consistent cross-reaction to OMP waspresent. We assume that the observations in the elderlypatients reflect extensive priorexposure to infecting uropatho-gens.As noted earlier, an elderly individual with bacteriuria and

fever without other signs or symptoms referable to thegenitourinary tract may present a difficult diagnostic prob-lem. Our observations suggest that antibodies to the majorOMP of E. coli correlate with invasive urinary tract infec-tion, showing a rise in titer in the convalescent phase. Inaddition, antibodies to the major OMP are widely cross-reactive (5). Thus, development of an assay to quantitate theantibody to major OMP may be useful as a serologic markerof invasive infection in this population.

ACKNOWLEDGMENTS

We thank D. Holton and P. Muir for assistance in collectingclinical specimens for this study and V. Svetina Atkins and B.Kowalczyk for secretarial support.This work was funded in part by a grant from the MSI Foundation

of Alberta, in part by an establishment grant from the AlbertaHeritage Foundation for Medical Research, and in part by theManitoba Medical Services Foundation.

LITERATURE CITED1. Boscia, J. A., W. D. Kobasa, R. A. Knight, E. Abrutyn, N. E.

Levison, and D. Kaye. 1986. Epidemiology of bacteriuria in anelderly ambulatory population. Am. J. Med. 80:208-214.

2. Esposito, A. L., R. A. Gleckman, S. Cram, M. Crowley, F.McCabe, and M. S. Drapkin. 1980. Community-acquired bacte-remia in the elderly: an analysis of one hundred consecutiveepisodes. J. Am. Geriatr. Soc. 28:315-319.

3. Hancock, R. E. W., and H. Naikaido. 1978. Outer membrane ofgram negative bacteria: XIX. Isolation from Pseudomonasaeruginosa PAD1 and use in reconstitution and definition of the

J. CLIN. MICROBIOL.

on June 25, 2020 by guesthttp://jcm

.asm.org/

Dow

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permeability barrier. J. Bacteriol. 136:381-390.4. Hansen, L. A., S. Ahlstedt, A. Fasth, U. Jodal, B. Kaiser, P.

Larsson, U. Lindberg, S. Olling, A. Dohl-Akerlund, and C.Svanberg-Eden. 1977. Antigens of Escherichia coli, humanimmune response, and the pathogenesis of urinary tract infec-tions. J. Infect. Dis. 136:144-149.

5. Hofstra, H., and J. Danhert. 1980. Major outer membraneproteins: common antigens in Enterobacteriaceae species. J.Gen. Microbiol. 119:123-131.

6. Holmgren, J., and J. W. Smith. 1975. Immunologic aspects ofurinary tract infections. Progr. Allergy 18:289-352.

7. Kaye, D. 1980. Urinary tract infections in the elderly. Bull. N.Y.Acad. Med. 56:209-220.

8. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

10. Nicolle, L. E., J. Bjornson, G. K. M. Harding, and J. A.MacDonell. 1983. Bacteriuria in elderly institutionalized men.

N. Engl. J. Med. 309:1420-1425.11. Nicolie, L. E., W. J. Mayhew, and L. Bryan. 1987. Prospective

randomized comparison of therapy and no therapy for asymp-tomatic bacteriuria in institutionalized elderly women. Am. J.Med. 83:27-33.

12. Nicolle, L. E., M. Mclntyre, H. Zacharias, and J. A. MacDonell.1984. Twelve month surveillance of infections in institutional-ized men. J. Am. Geriatr. Soc. 32:413-519.

13. Overbeehe, N., and B. Lugtenberg. 1980. Major outer membraneproteins of Escherichia coli strains of human origin. J. Gen.Microbiol. 121:373-380.

14. Shand, G. H., H. Anwar, J. Kadurugamuwa, M. R. W. Brown,S. H. Silverman, and J. Melling. 1985. In vivo evidence thatbacteria in urinary tract infection grow under iron-restrictedconditions. Infect. Immun. 48:35-39.

15. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

16. Weksler, M. E. 1981. The senescence of the immune system.Hosp. Pract. 16:53-64.

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