JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1987, p. 2181-21880095-1137/87/112181-08$02.00/0Copyright C 1987, American Society for Microbiology

Vol. 25, No. 11

Serum Antibodies to Outer Membrane Proteins of Escherichia coliin Healthy Persons and Patients with Bacteremia

ARNE Z. HENRIKSEN* AND JOHAN A. MAELAND

Department of Microbiology, Faclulty of Medicine, University of Trondheim, Trondheim 7006, Norway

Received 20 April 1987/Accepted 10 August 1987

Antibodies to Escherichia coli outer membrane proteins in sera from healthy persons and from patientsbacteremic with various enteric or nonenteric bacteria were measured by an enzyme-linked immunosorbentassay (ELISA). Outer membranes were prepared from E. coli 055. Serum was absorbed with E. coli 055lipopolysaccharide and diluted 1:100 for immunoglobulin A (IgA) or IgM and 1:1,000 for IgG antibodies.Paired serum specimens were obtained from the 56 patients included in the study (the first specimen on the dayof positive blood culture and the second specimen 8 to 12 days later) and compared with sera from blood donors(n = 50) as controls. On an average, the patients bacteremic with enterobacteria (n = 40) showed increasedlevels of antibodies of all three immunoglobulin classes in the first serum specimens and significantly higherlevels in the second specimens compared with the controls, although with considerable case-to-case variation.Increased levels of IgG antibodies showed the best cQmbination of diagnostic specificity (100%) and sensitivity(53%) for bacteremia caused by enteric bacilli. Mostly, the antibody response was directed against the majorE. coli 055 outer membrane proteins at 81,000, 38,500, 33,500, and 7,500 molecular weights as shown byWestern blot (immunoblot) analysis. Some of the patients bacteremic with nonenteric bacteria showedincreased levels of IgA antibodies, but not of IgG or IgM antibodies. Cross-reactivity of the nonenteric bloodculture isolates with the E. coli outer membrane preparation was not demonstrated. The cross-reactivity of theE. coli 055 outer membrane proteins with those of enteric bacilli of other genera was examined by absorptionexperiments. Western blots with serum absorbed with live E. coli 055 provided evidence that the epitopes ofthe outer membrane protein at 7,500 molecular weight were available for antibody binding at the bacterialsurface, and that at least some of the epitopes of the 38,500- and 33,500-molecular-weight proteins were

accessible to antibodies. The results suggest that an ELISA for the measurement of antibodies againstcross-reactive outer membrane proteins from enteric bacilli may be useful in the diagnosis of serious infectionscaused by members of the family Enterobacteriaceae, and that antibodies to the major outer membrane proteinsmay have an immunobiological function.

The serodiagnosis of infections caused by members of thefamily Enterobacteriaceae is hampered by the fact thatimportant antigens of these bacteria harbor antigenic deter-minants of variable specificities, such as the lipopolysaccha-ridé (LPS) and the capsular and flagellar antigens. Antigensof a nonprotein nature which are shared by the enteric bacilliinclude the lipid A moiety of LPS (9), determinants of Rforms of LPSs (29), and the common enterobacterial antigen(23). The levels of serum antibodies to these antigens inhealthy humans and in patients with infectious diseases havebeen reported previously (26, 28, 30, 37, 38, 45). However,serodiagnostic tests for antibodies against these antigenshave hot attained common use.Outer membrane (OM) proteins of the Enterobacteriaceae

harbor common epitopes (2, 3, 17, 19; A. Z. Henriksen andJ. A. Maeland, Acta Pathol. Microbiol. Immunol. Scand.Sect. B, in press). The cross-reacting OM proteins includethe porin protein, the heat-modifiable protein, and the lipo-protein, with molecular weights of 38,500, 33,500, and 7,500,respectively, as recently determined with OMs from anEscherichia coli 055 isolate (17; Henriksen and Maeland, inpress). OM proteins function as immnunogens in patients withinfectious diseases caused by different gram-negative bacte-ria (14-16, 27). Thus, in meningococcal disease, determina-tion of the levels of antibodies of the immunoglobulin G(IgG) class against meningococcal OM proteins verified theinfection in 84.5% of the patients (16). To our knowledge,

* Corresponding author.

only a few studies of human serum antibody levels againstenterobacterial OM proteins have been reported (1, 5, 12, 36,39). Either whole OM preparations (1, 36, 39) or single OMproteins (5, 12) were used as antigens in these studies, withfavorable diagnostic results in patients with typhoid fever (1,39) but with less favorable results in other patients withinfections caused by enteric bacilli (12).

In the present study, we measured levels of IgG, IgA, andIgM to OM proteins of an E. coli 055 strain in paired serumsamples from patients with bacteremia caused by variousenteric bacilli. The antibody levels were compared withthose of healthy individuals and patients infected bynonenteric bacteria. Experiments to elucidate some addi-tional aspects of the immune response by humans to entero-bacterial OM protein antigens are included.

MATERIALS AND METHODSPatients. A total of 56 hospitalized patients with bacte-

remia were tested. These included 40 patients (mean age, 71years) with bacteremia caused by a member of the Entero-bacteriaceae and 16 patients (mean age, 52 years) withbacteremia caused by nonenteric bacteria. BACTEC 6B and7D media were used for growth detection in blood culturewith an automated system (BACTEC 460; Johnston Labo-ratories, Inc., Towson, Md.). The numbers of enteric bloodculture isolates were as follows: E. coli, 26; Enterobacterspp., 2; Proteus spp., 5; Klebsiella spp., 4; Salmonella spp.,3. The numbers of nonenteric isolates were as follows:Streptococcus pneumoniae, 3; various streptococci, 4; Neis-

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2182 HENRIKSEN AND MAELAND

TABLE 1. Clinical data of patients with bacteremia

Blood culture isolate

Parameter Enteric Nonentericbacilli bacteria

No. of patients studied 40 16No. female/no. male 19/21 9/7Mean age (yr) 71 52No. with known focus of infection 30 ilNo. with septicemia 32 ilNo. without septicemia 8 5No. with acute leukemia 2 0No. on corticosteroid therapy 2 1Nô. with malignant tumor 8 3

seria meningitidis, 2; Haemophilhs infliuenzae serotype b, 2;Pseuidomonas spp., 2; Bacteroides fragilis, 2; Staphylococ-cus aureus, 1. Consecutive patients with positive bloodcultures were included in the study, irrespective of sex, age,origin of the bacteremia, or underlying disease. Sera werecollected from all the patients at the time of the bacteremiaand 8 to 12 days later and were stored at -20°C until tested.When the testing was finished, the clinical records of thepatients were checked. Table 1 summarizes some of theclinical data of the patients included in the study.

Controls. Sera from 50 blood donors (mean age, 39 years)and from 31 geriatric patients (mean age, 73 years), hospi-talized for degenerative joint diseases and without a historyof infectious disease for the last year, served as controls.Sera from children of various age groups were selected fromamong those forwarded to our department for serologicaltesting. The children suffered from various noninfectiousdiseases or from viral upper respiratory tract infections.

Bacteria. E. coli serotypes 014, 026, and 055, Neisseriagonorrhoeae NRL-8659, and N. meningitidis 44/76 sero-

group B were obtained as described previously (Henriksenand Maeland, in press). Other strains of bacteria used in theexperiments were from the collection of clinical isolates ofthis laboratory. These and the blood culture isolates frompatients under study were identified by biochemical meth-ods, supplemented by serological methods with strains forwhich antisera were available commercially. Generally, theculture media and identification methods were those recom-mended previously (43). The bacteria were stored in Stuarttransport medium at -80°C and were cultured and harvestedas described before (17).OMs. OMs were prepared from E. coli 055 by the method

described by Schnaitman (34, 35). The preparation wasfurther refined by washing with Tris hydrochloride (100mM)-EDTA (10 mM) buffer (pH 8.5) which contained 0.5%sodium deoxycholate (wt/vol) (17). The preparation, whichcontained the Triton X-100- and sodium deoxycholate-insoluble OM constituents, was finally washed with distilledwater and lyophilized.LPS. LPS was extracted from E. coli 055 by the method of

Westphal et al. (44), purified as described previously (17),and Iyophilized.

Antisera. Antisera against bacteria of E. coli 014, 026,and 055 were raised in rabbits as described before (17). Amixture of equal volumes of the E. coli 014 and 026 antiserais referred to as the heterologous E. coli antiserum. Purifiedantibodies to E. coli OM protein antigens (anti-OM) wereprepared from the heterologous E. coli antiserum by im-munoadsorbent chromatography (17), using a column of E.coli 055 OMs linked to epoxy-activated Sepharose 6B

(Pharmacia, Uppsala, Sweden). The anti-OM contained an-tibodies to the major E. coli 055 OM proteins, with molec-ular weights of 38,500, 33,500, and 7,500 (17; Henriksen andMaeland, in press).ELISA. An enzyme-linked immunosorbent assay (ELISA)

was carried out essentially as described by Voller et al. (42),using polystyrene microtiter plates (Nunc, Roskilde, Den-mark) and 50-p.l volumes of each of the reagents. Coats wereprepared with sonicated OMs (20 lg/ml), LPS (7.5 p.g/ml), orbacterial whole-cell sonicates (-107 bacteria per ml [17]).Before the ELISA, all human sera were absorbed by E. coli055 LPS (2 p.g of LPS per pi of serum). The mixture wasincubated at 4°C for 18 h and spun at 3,000 x g for 15 min,and the supernatant was tested for anti-E. coli OM antibod-ies. Human serum (1:100 or 1:1,000) or rabbit antiserumdiluted in phosphate-buffered saline (PBS) with 0.05%(vol/vol) Tween 20 (Henriksen and Maeland, in press) wastested in duplicate paired serum specimens on the samemicrotiter plate. Antibody binding was detected by alkalinephosphatase-conjugated goat antibody to rabbit IgG(1:1,000; Sigma Chemical Co., St. Louis, Mo.) when therabbit antibody was tested, and by alkaline phosphatase-conjugated goat antibodies to human IgG, IgA, or IgM(1:1,000; Sigma) when human sera were tested. At 45 minafter addition of the substrate, the color development wasrecorded at 405 nm (Microelisa Minireader MR 590; Dyna-tech Laboratories Inc., Alexandria, Va.) against controls inwhich the antigen was omitted from the coating buffer or theserum was replaced by PBS-Tween 20. Serum from a patientwith septicemia caused by E. coli was used as positivecontrol on each microtiter plate. The ELISA activity isexpressed as ELISA units calculated as optical density at405 nm x reciprocal of serum dilution x correction factor.The correction factor was calculated as follows: opticaldensity at 405 nm for positive control serum/mean opticaldensity at 405 nm for control serum (15 tests).SDS-PAGE and immunoblotting. OMs (20 or 50 ,ug) or

Iyophilized bacteria (50 ptg) were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25) as reported previously (17). The gels were eitherstained for proteins with Coomassie brilliant blue R-250 orsubjected to electrophoretic transfer of OM componentsonto nitrocellulose membranes as described before (17). Thenitrocellulose membrane strips were processed for immuno-blotting (Henriksen and Maeland, in press). Briefly, thestrips were incubated in 2% (wt/vol) bovine serum albuminin PBS for 15 min, then incubated (1 h) with antiserumdiluted in PBS-Tween 20 (0.05%), and washed with PBS.After incubation (1 h) with peroxidase-conjugated rabbitantibodies to human IgG, IgA, or IgM (1:1,000; Dakopatts,Glostrup, Denmark) or peroxidase-conjugated goat antibod-ies to rabbit lgG (1:1,000; Bio-Rad Laboratories, Richmond,Calif.), the color was developed as described before (31).Standard proteins were included for estimation of apparentmolecular weights (SDS-PAGE Standard Low MolecularWeight; Bio-Rad).

Absorption of serum with bacteria. Sediments of live E.coli 055 bacteria were used for absorption of 0.1 volume ofserum from a patient with septicemia caused by E. coli, afterdilution of the serum (1:100) in PBS-Tween 20. The mixturewas incubated at 20°C for 2 h and spun at 10,000 X g for 15min, and the supernatant was saved for analysis by ELISAand immunoblotting. Unabsorbed serum or serum absorbedin the same way with N. gonorrhoeae NRL-8659 was used asa control. The serum was also absorbed with Iyophilizedmaterial (1 mg/10 p1i of serum) of sonicated bacteria of

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SERUM ANTIBODIES TO E. COLI 2183

various species. Absorbed and unabsorbed sera were testedfor anti-OM antibodies (IgM) by ELISA.

Statistical analysis. Student's t test was employed forcomparison of means of ELISA units. We defined P < 0.01as being compatible with a significant difference betweenmeans. Indices of diagnostic reliability of the tests werecalculated (41) as follows: specificity = nondiseased personsnegative to the test/all nondiseased persons tested x 100;sensitivity = diseased persons with positive test/all diseasedpersons tested x 100; predictive value of positive test =number of diseased persons with positive test/total numberof persons with positive test x 100; predictive value ofnegative test = number of nondiseased persons with nega-tive test/total number of persons with negative test x 100.The group of nondiseased persons in the study included thehealthy controls and the patients with bacteremia caused bynonenteric bacilli.

RESULTS

Test system. Figure 1 shows the SDS-PAGE and im-munoblot patterns of the E. c(li 055 OM proteins used as anantigen in the ELISA. The major OM proteins, with molec-ular weights of 38,500, 33,500, and 7,500, demonstrateddistinct lines after staining with Coomassie brilliant blue andcorresponding lines on immunoblots, using the heterologousrabbit anti-E. coli serum (Fig. 1). With the homologousanti-E. coli serum, the same protein lines were seen inaddition to multiple lines rising from strain 055 LPS (17).The dilutions of the human sera used in the ELISA for IgA

or IgM anti-OM antibodies (1:100) or for IgG antibodies(1:1,000) were chosen on the basis of testing of variousdilutions of a few sera from patients and healthy individuals.Antibody production to the E. coli 055 LPS was not shownin any of the human sera tested. The patient serum used asa positive control in the ELISA for antibodies of the variousclasses of immunoglobulin never differed in optical densityby more than 10% of the mean values recorded on multipletesting of this serum.

Antibody activity in control sera. Sera from individuals ofvarious age groups and without infectious diseases causedby bacteria were tested by the ELISA (Table 2). IgA andIgM antibody activities against the E. (oli 055 OM proteinantigens were mostly low or not detectable. A much higherIgG antibody activity was measured. IgG anti-OM antibod-ies were present in every serum sample tested, although withconsiderable individual variation in the antibody levels. Forall three immunoglobulin classes, the average ELISA unitsshowed a gradual increase with increasing age of the chil-dren. The IgG levels reached those of the adults beforeadolescence. In the elders, antibody levels differed slightlyfrom those of the blood donors, but not significantly (P >0.5).We defined a positive test as greater than or equal to the

mean plus 3 standard deviations of the ELISA units re-corded with sera from the blood donors. No serum in thecontrol groups showed a positive test for antibodies of any ofthe three immunoglobulin classes. If we defined a positivetest as greater than or equal to the mean plus 2 standarddeviations, then 6% of the controls would show a positivetest for IgG and IgA antibodies, and 4% would be positivefor IgM antibodies.

Antibody levels in patients with bacteremia. Figure 2 showsthe ELISA units for each of the blood donors and each of thebacteremia cases. For all three immunoglobulin classes. thecase-to-case variation in antibody levels was particularly

81 k __

38.5 k-MJ33.5 k-amp

1 7.5 k-,o__

7.5 k-

FIG. 1. OMs from E. coli 055 separated by SDS-PAGE andstained for proteins (50 ,ug; left lane) or analyzed by immunoblotting(20 p.g; right lane). using heterologous anti-E. coli serum (1:1,000).

marked for enterobacterial bacteremia. For instance, for IgGantibodies, the coefficients of variation were 55% for theblood donors, and 81.7 and 60.6% for the second serumsamples for the bacteremia cases caused by enteric andnonenteric bacteria, respectively. The patients with bactere-mia caused by the Enterobacteriaceae showed increasedmeans of ELISA units for anti-OM antibodies of all threeimmunoglobulin classes in the first serum sample (Fig. 2;Table 3). For IgG and IgA antibodies, the difference wassignificant (P < 0.001) when matched against the controls.With the second serum samples, further increases in levelsof antibodies were recorded. The levels significantly ex-ceeded those of the controls for all three classes (P < 0.001)and those of the first serum samples for IgG and IgMantibodies (P < 0.01), but not for IgA antibodies.

For the patients bacteremic with enteric bacilli, the fre-quency of positive tests for IgM, IgG, and IgA antibodiesincreased in that order, and was from two to three timeshigher with the second serum sample than with the firstsample (Table 3). Approximately 80% of these patientsshowed an increase in ELISA units from the first to thesecond samples. Doubling or higher increments of ELISAunits for IgG, IgA, and IgM antibodies were recorded for 37,27, and 32%c of the patients, respectively (Table 4). Some ofthe patients with a positive test in the first sample showedunchanged or slightly reduced antibody levels in the secondsample.

Patients bacteremic with E. (oli (26 patients) and thosebacteremic with other enterobacteria (14 patients) showedessentially the same antibody response (data not shown).The patients septicemic with enteric bacilli (n = 32) showeda stronger antibody response with all three immunoglobulinclasses than did those with bacteremia without septicemia (n= 8). Thus, with the second serum sample, 20 (62.5%) of thesepticemic patients showed a positive IgG antibody test,whereas only 1 (12.5%7) of those without septicemia showeda positive test (0.02 > P > 0.01: x2 test). The septicemicgroup included two patients with acute leukemia, none of

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2184 HENRIKSEN AND MAELAND

TABLE 2. Antibodies to E. coli 055 OM proteins in sera from individuals of various age groups andwithout infection caused by bacteria

Mean ELISA units ± SD (range)':Age group (yr) n

IgG IgA IgM

Children<1 12 150 ± 112 (40-390) 4.2 ± 4.1 (0-12) 4.8 ± 4.3 (0-15)1-5 12 155 ± 106 (30-370) 4.8 ± 2.8 (0-7) 17.8 ± 10.3 (1-35)6-9 13 250 ± 130 (30-400) 5.5 ± 3.7 (0-11) 27.6 +10.8 (5-40)10-16 il 266 ± 230 (60-570) 10.0 + 3.4 (3-14) 28.6 +15.1 (6-42)

AdultsBlood donors (mean age, 39) 50 222 ± 123 (50-540) 7.8 ± 5.2 (0-22) 17.4 + 8.1 (3-41)Elders (mean age, 73) 31 172 ± 149 (10-560) 11.7 ± 7.9 (0-30) 11.0 + 6 1 (1-29)a Optical density at 405 nm x reciprocal of serum dilution x correction factor (see the text).

which showed an anti-OM antibody response. An antibodyresponse was shown by both of two patients in this group onimmunosuppressive therapy after renal transplantation.Some of the patients (n = 16) with bacteremia caused by

nonenteric bacteria responded with elevated levels of IgAantibodies against the E. coli 055 OMs (Fig. 2; Table 3).Three of them showed a positive test with the first serumsamples and further three with the second samples, four ofwhich had septicemia and two of which had bacteremiawithout septicemia. None of them showed rising levels ofIgG or IgM antibodies.The data shown in Table 5 describe the serodiagnostic

value of the ELISA in infections caused by the Enterobac-teriaceae, as recorded with the second serum samples. Apositive test for IgG antibodies showed 100% specificity andpredictive value, but with a sensitivity of only 53%. Thereliability of a positive test for IgM antibodies was equal tothat for IgG, but with even lower sensitivity (40%). Elevatedlevels of IgA antibodies showed the highest sensitivity, butthe lowest specificity, for enterobacterial bacteremia.

Further analysis of anti-E. coli OM antibodies. Rising levelsof IgA antibodies against the E. coli 055 OMs in some of thepatients bacteremic with nonenteric bacteria incited experi-ments to test for cross-reactivity of these bacteria with theOM preparation. ELISA coats were prepared with ultrason-ically disrupted bacteria of all of the nonenteric blood cultureisolates. None of the coats showed binding of affinity-purified rabbit antibodies to the E. coli 055 OM proteinantigens. Whole-cell SDS lysates of the same nonentericbacteria were separated by SDS-PAGE and transblottedonto nitrocellulose membranes for immunoblotting. Theaffinity-purified anti-OM (1:100) showed no antibody bindingto electroblotted materials from these bacteria. Distinct lines(Fig. 1) were seen in Western blot (immunoblot) analysiswith the E. coli 055 OMs. These results argue against thehypothesis that elevated levels of IgA antibodies in thepatients with bacteremia caused by nonenteric bacteria weredue to antigens in these bacteria which cross-reacted withthose of the E. coli 055 OMs.

Sera from five of the blood donors and from nine of thepatients with bacteremia caused by various genera of theehteric bacilli were selected on the basis of ELISA activityand tested by Western blotting for IgG, IgA, and IgMantibodies against proteins of the E. coli 055 OMs. With theblood donor sera, faint bands appeared that were caused byIgG antibodies against one or more of the OM proteins withmolecular weights of 38,500, 33,500, and 7,500. Sera fromthe patients gave rise to stronger lines corresponding to oneor more of the same OM proteins and, in addition, lines

A

IgG1800

1500

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900

8600

300

IgA

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C

c

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41

B

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'I' T.150

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50 ~

O t w ïI *

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Control 1. sample 2. sample 1. sample 2. sample(n=50) (n=40) (n =16)

FIG. 2. ELISA units for IgG, IgA, and IgM antibodies against E.coli 055 OM proteins in serum from blood donors (A; n = 50) andin paired serum specimens from patients with bacteremia caused byenteric bacilli (B; n = 40) or varioUs nonenteric bacteria (C; n = 16).The long horizontal lines indicate the mean plus 3 standard devia-tions of ELISA units for the blood donors, and short horizontal linesindicate means of ELISA units.

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SERUM ANTlBODIES TO E. COLI 2185

TABLE 3. Antibodies to E. coli 055 OM proteins in paired serum samples from patients with bacteremia

ELISA units in sample:

No. of Immunoglobulin 1patients class

Mean + SD Range Mean ± SD Range(%4 positive) (% positive)

Enteric bacilli 40" G 385 + 311 (22)' 40-1.400 574 + 469 (53)" 60-1,990A 25.6 + 28.8 (35)"' 2-124 35.4 + 34.6 (65)" 2-150M 22.9 + 19 (12) 1-94 40.0 + 33.6 (40)h 5-132

Nonenteric bacteria 16' G 167 + 116 (0) 50-470 165 + 100 (0) 50-390A 15.9 + 15.9 (19)" 4-62 21 + 16.5 (37)" 457M 13.1 + 7.2 (0) 0-33 16.3 + 9.5 (0) 2-33

Mean age, 71 vears."P < 0.01 when compared with controls (sec Table 2).Mean age. 52 years.

corresponding to one or both of the OM proteins withmolecular weights of 81,000 and 17,500 with some of thesera. This was the case for antibodies of ail three immuno-globulin classes, in agreement with the ELISA results forclass-specific antibodies. In general, the staining intensitydue to antibody binding by the electroblotted OM proteinswas stronger with the second serum samples than with thefirst samples. However, case-to-case variation of the pro-

teins that incited the antibody response was noted. Figure 3shows an example with serum from a patient who developedan antibody response mostly against the E. coli OM proteinof 33,500 molecular weight and, to a lesser extent, againstother major OM proteins.

Experiments were performed to test the human serum

antibodies which combined with the E. coli 055 OM proteinsfor reactivity with antigens in bacterial sonicates of one

strain of each of the following species: Citrobacterfreundii.Enterobacter cloacaie, E. coli 026, Klebsiella pneuinoniae,

Proteus v'ulgaris, Salmonella ininnesota, and Yersinia en-

terocolitica 03. ELISA activity of the second serum samplefrom a patient septicemic with E. coli was reduced in therange of 65% (Y. enterocolitica) to 94% (C. fiJeundii) by theabsorptions. Similar absorptions performed with sonicatedbacteria of H. influenz-ae serotype b. N. mneningitidisserogroup B, or Pseudotnonas aeruginosa resulted in lessthan 10% reduction of the optical density readings. Theresults agreed with cross-reactivities of human serum anti-bodies against the E. coli 055 OM proteins with antigens ofthe other enterobacteria tested, presumably the OM proteinsof these bacteria.A second serum sample from the same patient was also

absorbed with live E. coli 055 bacteria and then tested. Theabsorption resulted in 70% reduction of the ELISA activityand, as shown by immunoblotting, in loss of the ability toinduce a line corresponding to the OM protein of 7,500molecular weight. Also, a strong reduction in the antibody-

TABLE 4. Increments of ELISA units of antibodies to E. coli055 OM proteins in 40 patients with bacteremia

caused by enteric bacilli

Immunoglobulin <% of patients with ELISA unit increments':

class 1 1-2 2-3 3-4 >4

G 20 43 15 12 10

A 25 48 15 2 10M 20 48 12 5 15

ELISA units of second serum sample/ELISA units or first serum sample.

mediated staining intensity of the protein line at 33,500 andsome reduction of the protein line at 38,500 molecular weightwere observed. The results were indecisive regarding theprotein fine at 81,000 molecular weight. Similar absorptionsperformed with live N. gonorrhoeae did not affect theELISA activity or the antibody-mediated staining intensityof any of the major E. (oli 055 OM protein lines.

DISCUSSION

Enterobacterial OM proteins have been extensively char-acterized (4, 10, 13, 32), including the demonstration ofimmunogenicity in experimental animals (3, 8, 18, 19).Reports by others (5, 12, 39), as well as the data described inthe present study, show that OM proteins of these bacteriaincite an antibody response in patients with infectionscaused by the Enterobaccteriaceae. This observation agreeswith findings in patients with infections caused by variousnonenteric gram-negative bacteria (14-16, 27, 40). That theserum antibodies measured by us were directed againstproteins of E. c oli 055 OMs was substantiated by thefindings that all sera tested were negative in ELISA with theE. coli 055 LPS coat, presumably because of absorptionwith the LPS, and that the class-specific antibody activityagainst OM proteins as observed by Western blot analysiscorresponded to that recorded by the ELISA.From shortly after birth, the human intestinal tract is

colonized by bacteria which include enterobacteria (33).This probably explains our observation that children evenbelow 1 year of age possessed serum antibodies againstproteins of the E. ( oli 055 OMs, notably IgG antibodies, andthat the levels of the anti-OM antibodies increased duringchildhood. The results agree with the observations (13) thatsera from humans, including children, frequently contain

TABLE 5. Indices of validity and reliability in the serodiagnosisof bacteremia caused by enteric bacilli"

%E for immunoglobulin class:Parameter

G A M

Sensitivity 53 65 40

Specificity 100 91 100

Predictive value of test:Positive 100 82 100Negative 78 81 73

ELISA of second serum specimens with E. coli 055 OM protein antigens.

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.. 2

Igm~~~~~~~~~~~~~~~~~~~~~~g:"IgA

FIG. 3. First (left lanes) and second (right lanes) serum samplesfrom a patient septicemic with E. coli analyzed by immunoblottingfor IgG (1:1,000), IgM, and IgA antibodies (1:100) against E. coli055 OM proteins.

IgG antibodies against the iron-regulated E. coli OM proteinsand against the OmpA protein, which corresponds to the33,500-molecular-weight protein of the E. coli 055 OM.These data support the concept that serum antibodies to OMproteins of the enterobacteria are ubiquitous in the popula-tion, although with considerable individual differences inantibody levels.When compared with the controls, the patients with

enterobacterial bacteremia, on average, showed elevatedlevels of anti-OM protein antibodies of all three immuno-globulin classes at the time when the bacteremia was estab-lished, some with peak antibody activity at that time. This issimilar to observations of children with meningitis caused byH. influenza (40). Our observations can be explained byassuming that the bacterium elicited an antibody responseagainst the OM proteins through inapparent infection or alocalized infectious process before the bacteremia, or that insome of the patients bacteremia had lasted for some timebefore it was verified. We found that IgA antibodies showedparticularly swift rises. This may be linked with the normalassociation of enteric bacilli with mucosal surfaces. Similarfindings were recorded in patients with infections caused byother gram-negative bacteria which normally associate withmucosal surfaces (22). A swift and strong IgA antibodyresponse may adversely affect the susceptibility of individ-uals to invasive bacteria (11).The diagnostic sensitivity and specificity of the antibody

determinations differed from one class of antibody to an-

other, as has also been pointed out by other investigators(16, 22). However, we cannot exclude that. in the currentstudy, the IgA and IgM antibody levels were underestimatedbecause of competition from the IgG anti-OM antibodies.Our data indicate that anti-OM IgG and IgM antibody risesmay be specific for infections caused by enteric bacilli. IgAantibody determinations showed lesser specificity. This in-ference is related to the observation that IgA anti-OMantibody levels increased in some of the patients withbacteremia caused by nonenteric bacteria. Since none of thenonenteric blood culture isolates showed cross-reactivitywith the E. coli OM proteins, we infer that the antibody risesthrough nonspecific mitogenic stimulation of cell cloneswhich produce IgA antibodies to enterobacterial OM pro-teins. It appears that an increase in antibodies (including

autoantibodies) caused by nonspecific stimulation occursfrequently in humans with serious infections caused bybacteria (21, 22, 46), and in rabbits immunized with gram-negative bacteria (20). Why the nonspecific stimulus mostlyaffects IgA-producing cell clones is not known, but it may berelated to the mucosal route of stimulation by the Entero-bacteriaceae in normal situations.Of the three immunoglobulin classes, increased levels of

IgG antibodies against the E. coli 055 OM proteins showedthe best combination of diagnostic specificity and sensitivityfor enterobacterial bacteremias. However, the sensitivityrose to only 22 and 53% with the first and second serumspecimens, respectively, using a cutoff as defined in thisstudy. Increments of ELISA units for IgG anti-OM antibod-ies were observed for 80% of the patients in the experimentalgroup, but only 37% of them showed doubling or more fromthe first to the second serum sample. Significantly elevatedlevels of IgG antibodies or increments corresponding to atleast doubling of the ELISA units, when taken together,were shown by 62% of the patients.The diagnostic sensitivity of the IgG antibody determina-

tion as recorded in this study was superior to that of indirecthemagglutination testing of antibodies against the E. colilipoprotein (12) but was inferior to the ELISA for antibodiesagainst Salmonella typhi OM porin proteins (5) or a radio-immunoassay for antibodies against the cell envelope frac-tions in patients with typhoid fever (39). The discrepancymay be explained on the basis of differences in the route ofantigen presentation, the intensity of immunostimulationduring infection caused by different enterobacteria, theduration of the illness, or in the selection of the antigen foruse in the antibody testing. The ELISA for enterobacterialinfection was also inferior in sensitivity to that of ELISA formeningococcal disease, in which a sensitivity of 84.5% wasrecorded (16). The majority of the patients with meningococ-cal disease were children or adolescents without underlyingdisease, whereas those of the present study mostly wereelders, some of them with serious underlying disease, whichmay explain the discrepancy. On the other hand, the pro-pensity of elders to develop an antibody response is com-paratively well preserved (6).

For antigens in a diagnostic test it would be impractical touse OM proteins from bacteria of a variety of enterobacterialspecies. We observed that patients septicemic with bacteriaof different species of the Enterobacteriaceae developedantibodies which combined with the E. coli 055 OM pro-teins, and that antibodies to these proteins were removed toa considerable extent on absorption of serum from a patientsepticemic with E. coli with various enterobacteria. Theseresults substantiate earlier observations with rabbit antibod-ies, i.e., that enterobacterial OM proteins harbor commonepitopes (17; Henriksen and Maeland, in press). Thus, OMprotein antigens from one enterobacterial species, such as E.coli, when used in a serodiagnostic test should discloseantibody rises induced by ongoing infection caused bybacteria of the same species and by bacteria of other speciesof the Enterobacteriaceae. The Western blot analysis re-vealed individual variation with regard to selection of thevarious OM proteins as an immunogen, in addition to theindividual variation in the overall anti-OM antibody re-sponse (Fig. 2; Table 3). This would favor the use of OMprotein mixtures as an antigen in serodiagnosis, such as theOM preparation used in this study, rather than a single OMprotein antigen.

In spite of adequate specificity of the anti-E. coli OMELISA for enterobacterial bacteremias, the test showed

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SERUM ANTIBODIES TO E. COLI 2187

comparatively low sensitivity for unselected bacteremias.This may impose limitations to the clinical usefulness of thetest. Although the number of patients we have studied is notbig enough to permit firm conclusions, the results are inaccord with the tentative inferences that (i) seriously im-munocompromised patients will not respond with antibodyformation against the OM proteins, that (ii) the majority ofpatients with enterobacterial bacteremia without signs ofsepticemia will not show a positive test, and that (iii) themajority of immunocompetent patients with bacteremia andclinical signs of septicemia will develop a positive test. Thus,the anti-OM ELISA may be a tool to discriminate betweensevere and less-severe bacteremias. It remains for furtherinvestigations to substantiate whether the anti-OM ELISAwill be of prognostic value and whether the test has otherpotential applications, such as serological verification ofstrictly localized enterobacterial infections, assessment ofthe efficacy of antimicrobial therapy, or verification of theinfection when ongoing therapy interferes with isolation ofthe bacterium. With the anti-E. coli OM ELISA, testing ofserum drawn at intervals to disclose anti-OM antibodyincrease may be more useful than testing of single serumspecimens (Tables 3 and 4).

Evidently, the antibodies to the OM antigens did notprotect the patients from contracting bacteremia by theenteric bacilli in spite of comparatively high antibody levelsin some of the patients at the time when bacteremia wasverified. Thus, the protective capacity of these antibodies, ifany, was insufficient for combating the invasive microorgan-isms. Evidence for an immunoprotective function of theantibodies to OM proteins has come from results obtained inexperimental salmonellosis of mice (24) and from experimen-tal H. influenza infection (7). The results of the absorptionexperiment performed by us support the notion that epitopesof the OM protein of 7,500 molecular weight and at leastsome of the epitopes of the OM proteins with molecularweights of 38,500 and 33,500, when present in live E. coli055, were accessible for binding ofhuman serum antibodies.These observations support the possibility of in vivo im-munobiological functions of antibodies to enterobacterialOM proteins. For instance, it is tempting to speculatewhether antibodies to OM proteins participate in the im-munoprotection afforded by sera from humans immunizedwith heat-killed E. coli J5 (47). Experiments with monoclo-nal antibodies against the E. coli OM proteins are in progressin our laboratory to elucidate these possibilities.

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