INFECTION AND IMMUNITY, JUlY 1987, p. 1600-16060019-9567/87/071600-07$02.00/0Copyright C 1987, American Society for Microbiology

Antigenic Analysis of Serratia marcescens Fimbriae withMonoclonal Antibodies

SEIYA JINGUSHI,* MASAO MITSUYAMA, TETSUHIRO MORIYA, AND KAZUNOBU AMAKODepartment of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka, 812, Japan

Received 30 October 1986/Accepted 27 March 1987

Monoclonal antibodies (MAbs) were raised against the purified fimbriae of Serratia marcescens US46, a

strain expressing three morphologically distinct fimbriae. The widths of these fimbriae were 7, 4.5, and 3 nm,respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed threebands with molecular weights of 21,000, 20,000, and 19,000, respectively. This strain had mannose-resistant(MR) hemagglutinating activity and was agglutinated by yeast cells. Therefore, strain US46 appeared to haveboth MR and mannose-sensitive fimbriae. In the immunoblot analysis, all MAbs reacted with the 20,000-molecular-weight subunit when given a choice of three differently sized subunits. Immunoelectron microscopyshowed these MAbs attached to the MR fimbriae with the largest width (7 nm). The antigenic cross-reactivityof fimbriae was examined by an MAb-mediated agglutination test. All MR strains of S. marcescens and some

mannose-sensitive strains were agglutinated by the MAbs. The serological homogeneity of MR fimbriae was

confirmed by a spot test, using the crude purified fimbriae from several MR strains of S. marcescens. In othergram-negative rods, clinical isolates of Klebsiella spp. with hemagglutinating activity were agglutinated, butclinical isolates of Escherichia coli and Enterobacter spp. were not.

Fimbriae are filamentous appendages mostly prevalentamong gram-negative bacteria. They mediate the adherenceof the bacteria to host tissue surfaces. Fimbriae appear to beattractive candidates for vaccines that would prevent infec-tion at a very early stage (18, 19, 21, 31). However, it hasbeen reported that fimbriae exhibit a considerable degree ofinterstrain antigenic heterogeneity in various species ofbacteria (10, 14, 22, 25, 26, 28, 33). In uropathogenicEscherichia coli, 0rskov and 0rskov (25) distinguished eightserologically different fimbriae, and Parry et al. (28) foundseven serologically different fimbriae. Even among fimbriaeof the same group, such as K88 (10, 26), there is an antigenicdiversity. In addition to intra- and interstrain differences, a

single isolate can also undergo extensive antigenic variation.Variants of the P9 strain of Neisseria gonorrhoeae expressedone of four serologically different fimbriae (33), and variantsof a strain isolated from different anatomical locations ex-

pressed antigenically distinct fimbriae (35).Serratia marcescens is one species that may cause noso-

comial respiratory or urinary tract infections, and this spe-cies also expresses fimbriae (1). The adherence of S. mar-

cescens US5 to human bladder epithelial cells is also medi-ated by these structures (34). Therefore, these fimbriaemight be the source of an effective vaccine against infectionscaused by S. marcescens. In this respect, it is important toanalyze the antigenicities of the fimbriae expressed by vari-ous strains of S. marcescens.

We examined the antigenic cross-reactivity of S. marces-

cens fimbriae using monoclonal antifimbria antibodies andfound that the serological interstrain homogeneity of themannose-resistant (MR) fimbriae of S. marcescens was

remarkably high in contrast to that of the fimbriae of theother bacterial species.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains and culture conditions. All strains of S.marcescens, E. coli, Klebsiella spp., and Enterobacter spp.used in this study were clinical isolates obtained at FukuokaUniversity and Kyushu University Hospitals, Fukuoka,Japan. In S. marcescens strains, US series and SS serieswere isolated from patients with urinary tract and respira-tory tract infections, respectively. Strain US46 was used formonoclonal antibody (MAb) production. E. coli strainshaving colonization factor antigen I (CFAI) or CFAII fim-briae were obtained from Toshio Miwatani (Research Insti-tute for Microbial Diseases, Osaka University), and strainswith F7, F8, F9, or FEl fimbriae were from Riichi Sakazaki(National Institute of Health, Tokyo, Japan). These strainswere maintained on nutrient agar. For the purification offimbriae, the strains were cultured in CFA broth (5).

Purification of fimbriae. Fimbriae were purified as de-scribed by Honda et al. (13) with some modifications.Bacteria were cultured aerobically in CFA broth at 37°Covernight and were harvested by centrifugation at 10,000 x

g for 30 min. The pellet was suspended in 0.05 M Trishydrochloride buffer (pH 8.0) and homogenized with a

Waring blender (Nihonseiki, Tokyo, Japan), and thehomogenate was centrifuged at 25,000 x g for 60 min. Thesupernatant was concentrated to 1/10th of the starting vol-ume in a PM-10 membrane (Amicon Corp., Lexington,Mass.). This concentrate was used as the crude fimbriae inthe spot test. Crude fimbriae were further purified through a

Sepharose 4B column (Pharmacia, Uppsala, Sweden). Elu-tion of the fimbriae in the void volume was confirmed byelectron microscopy, and this fraction served as the purifiedfimbriae.MAb production. BALB/c mice were immunized subcuta-

neously with the purified fimbriae (40 jig of protein) inFreund complete adjuvant. A booster injection in Freund

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incomplete adjuvant was administered 2 weeks later. After 2weeks the animals were injected intravenously with the same

dose of fimbriae. Three days after the last injection, theanimals were killed, and their spleens were removed. Spleencells were fused with P3U1 myeloma cells (an azaguanine-resistant, nonsecreting line derived from BALB/c mice) andcloned as described by Ham et al. (12). Culture supernatantswere screened for antibodies by an enzyme-linked immuno-sorbent assay (ELISA). Hybridoma cells were cultured inDulbecco modified Eagle medium (GIBCO Laboratories,Chagrin Falls, Ohio) supplemented with 10% (vol/vol)Nuserum (Collaborative Research Inc., Waltham, Mass.),2.5 mM L-glutamine, 1 mM cis-oxaloacetic acid (SigmaChemical Co., St. Louis, Mo.), 0.4 mM sodium pyruvate(Sigma), 0.16 U of bovine insulin (Sigma) per ml, and 50 ,ugof gentamicin per ml. MAbs were purified from culturesupernatant by precipitation with 50% saturated ammoniumsulfate. The precipitate was suspended in sterile distilledwater and dialyzed against phosphate-buffered saline (PBS).In the immunoblot analysis, hybridoma cells were expandedin the peritoneal cavity of BALB/c mice, and the ascites wasused as the MAb supernatant after purification on a proteinA-Sepharose CL-4B (Pharmacia) column. To determine theisotypes of MAbs, MAb identification kits (Zymed Labora-tories, Inc., San Francisco, Calif.) were used.ELISA. Wells of microtiter plates were coated overnight at

4°C with 50 ,ul of a fimbria solution (5 ,ug/ml) per well incoating buffer (pH 9.6) and were washed three times withPBS-0.05% Tween 20 (PBST). Supernatants (50 ,ul) of eachhybridoma clone were added to the wells, and the prepara-tions were incubated for 90 min at room temperature. Forantibody titration and specificity testing, the wells were

incubated with serial dilutions of MAbs in PBST. Subse-quently, the wells were washed three times with PBST,followed by incubation with goat anti-mouse immunoglobu-lin conjugated with peroxidase (Tago Inc., Burlingame,Calif.) at room temperature. Antifimbria antibody activitywas detected colorimetrically by adding the peroxidasesubstrate o-phenylenediamine (Wako Pure Chemical Indus-tries, Ltd., Osaka, Japan). The A490 was measured with a

MicroELISA minireader (MR 590; Dynatech Laboratories,Inc., Alexandria, Va.).

Hemagglutination test. Chicken, guinea pig, sheep, andhuman type A erythrocytes (RBCs) were used for hemag-glutination tests as 2% suspension in PBS. A loopful from a

colony from a bacterial lawn on a CFA agar plate was

suspended in PBS with or without 1% (wt/vol) mannose on

glass slides. Erythrocytes were added to this suspension,and agglutination was determined within a few minutes (6,23).MAb-mediated bacterial agglutination test. A bacterial

suspension in PBS was mixed with MAbs on glass slides.Agglutination was determined within a few minutes. Thestrains which spontaneously agglutinated when suspended inPBS were excluded from the study.

Yeast cell agglutination test. Dry bakers' yeast (Sac-charomyces cerevisiae) (Nissin Flour Milling Corp., Tokyo,Japan) was suspended in PBS. The yeast cell suspension(2%, wt/vol) and the bacterial suspension were mixed on a

glass slide, and agglutination was assessed within a fewminutes (20).Immunoelectron microscopy. (i) Labeling of whole bacteria.

Colloidal gold was prepared by the method of Frens (9).Protein A-gold complex was prepared by the method of Rothet al. (29) with some modifications. Colloidal gold was addedto 2 mg of protein A (Pharmacia) in 0.25 M Tris hydrochlo-

ride buffer (pH 6.6) with shaking for 7 min at room temper-ature. Gold-protein A probe was collected by centrifugationat 20,000 x g for 30 min and suspended in 0.1% bovineserum albumin-PBS. For labeling the bacterial cells, bacte-ria were cultured in CFA broth, washed three times with 2%ammonium acetate, and incubated with MAbs at 37°C for 2h. After being washed with ammonium acetate, the protein-gold probe was added to the suspension. To remove theexcess unreacted probe, we washed the bacterial suspensionrepeatedly with ammonium acetate. The labeled specimenswere negatively stained with 2% sodium phosphotungstate(pH 7.0) and examined with a JEM 100C electron micro-scope at 80 kV.

(ii) Labeling of purified fimbriae. The purified fimbriaewere incubated with MAbs at room temperature for 2 h. Theagglutinate was washed three times with 2% (wt/vol) ammo-nium acetate and then incubated with goat anti-mouse im-munoglobulin antibodies conjugated with colloidal gold(Janssen Life Sciences Products, Div. Janssen Pharmaceu-tica, Beerse, Belgium) at room temperature for 2 h. Theremainder of the procedure was as described above.

(iii) Labeling of MR fimbriae on RBCs. The purified fim-briae were suspended in PBS containing 1% (wt/vol) man-nose (PBSM) and incubated for 16 h at 4°C. Chicken RBCs(2%, vol/vol) were added to this fimbrial suspension andwere incubated for 2 h at room temperature with gentleshaking to allow the fimbriae to adhere to the RBCs. Afterthe RBCs were washed twice with PBSM, MAbs againstUS46 fimbriae were added, and the mixture was incubatedfor 1 h at room temperature. The MR fimbriae that reactedwith the MABs on the chicken RBCs were labeled withprotein A-colloidal gold as described above.SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) was performed as described byLaemmli (17). The purified fimbriae were suspended in waterwhich had been adjusted to pH 1.5 with HCl, and they werethen boiled in a water bath for 5 min. After the mixturecooled at room temperature, the pH was neutralized with0.125 M NaOH. The depolymerized sample was applied to anongradient separating gel (15% acrylamide) with a 5%stacking gel and then electrophoresed at 20 mA of constantcurrent per slab. For calibration of the molecular weight, alow-molecular-weight marker kit (LKB Corp., Bromma,Sweden) was used.Immunoblotting analysis. After electrophoresis, the pro-

teins in the gel were transferred to nitrocellulose paper withan electroblot apparatus (Bio-Rad Laboratories, Richmond,Calif.) by the method of Towbin et al. (32). The residualprotein-binding sites on the nitrocellulose paper wereblocked by incubating it in PBST containing 1% (wt/vol)bovine serum albumin for 30 min. The blocked paper wasincubated in the MAb solution for 90 min at room tempera-ture. After three washings with PBST, the paper was incu-bated with 1:800-diluted goat anti-mouse immunoglobulin G(IgG)-peroxidase conjugate in PBST for 90 min at roomtemperature. Immune complexes were detected by adding0.4 mg of 4-chloro-1-naphthol (Nakarai Chemicals, Ltd.,Kyoto, Japan) per ml in 50 mM Tris buffer (pH 7.5) contain-ing 0.01% H202. The reaction was halted by dipping thepaper into distilled water.

Spot test for reaction of MAbs with fimbriae. Spot testsused in this study were a modification of the procedure of deRee et al (2). The suspensions of crude fimbriae obtainedfrom various strains of S. marcescens were spotted ontonitrocellulose paper and dried at room temperature. Theprocedure for determining reactivity with MAbs was the

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1602 JINGUSHI ET AL.

A B

US46

C

US5FIG. 1. Electron micrographs of negatively stained purified fimbriae from S. marcescens US46 and US5. The purified fimbriae from strain

US46 are a mixture of three different types with widths of 7 (A), 4.5 (B), and 3 (C) nm. The smallest fimbriae (C) resemble those of strain US5.

same as that described above for the immunoblotting analy-sis.

RESULTSCharacterization of strain US46 fimbriae. Examination of

the purified fimbriae of S. marcescens US46 by electronmicroscopy revealed the existence of three morphologicallydistinct types of fimbriae (Fig. 1). The widths of the threedifferent fimbriae were 7, 4.5, and 3 nm, respectively.Among these, the 3-nm fimbriae resembled those of strainUS5 which is known to have one type of mannose-sensitive(MS) fimbriae (15). In SDS-PAGE of the purified fimbriae ofstrain US46, six extensively stained peptide bands of dif-ferent molecular sizes were observed (Fig. 2). Three of thesemigrated at about 20,000 daltons, and the rest migrated atabout 40,000 daltons. The molecular weights of the first threebands, presumably monomers, were 21,000, 20,000, and19,000, respectively. The three bands with molecularweights ranging from 38,000 to 44,000 seemed to be dimersformed by each of the three bands appearing in the lowerportion of the gel. The band with the lowest molecularweight (19,000) was identical to that of the fimbrial subunitsof strain US5 (14). Although strain US46 showed MRhemagglutination with chicken RBCs, it also agglutinatedyeast cells, which are generally used to demonstrate thepresence of MS fimbriae (16, 19). These observations indi-cated that strain US46 had both MR and MS fimbriae. Itseemed likely that the 3-nm fimbriae are the MS fimbriae.

Reactivity and specificity of MAbs for fimbriae. ThreeMAbs against the fimbriae of strain US46 were obtained,

with the isotypes being two IgG2b and one IgM. Thespecificity of MAbs for the fimbriae was tested by thebacterial agglutination test, ELISA, immunoblot analysis,and immunoelectron microscopy. The reactivity of MAbs inthe bacterial agglutination test and the ELISA is shown inTable 1. The titers determined by bacterial agglutination and

A B C94 K

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FIG. 2. SDS-PAGE of purified fimbriae from S. marcescensUS46 (A) and US5 (B). Three different-sized subunits, presumablymonomers, of fimbriae from strain US46 and their respective dimersare shown by dots. MAb DH2 reacted with only the 20,000-molecular-weight subunit when given a choice of the purifiedfimbriae of strain US46 in the immunoblot analysis (C). K, 103.

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TABLE 1. Characterization of MAbs against the fimbriaeof strain US46

MAb Isotypea Bacterial ELISA titercagglutination titerb US46 USS

DH2 IgG2b 64 2,560 <40DC3 IgG2b 32 2,560 <40EH6 IgM 64 5,120 <40

a Antibody isotype was determined with rabbit anti-mouse isotype-specificantibodies in ELISA.

b Bacterial agglutination titers are expressed as the highest dilution ofMAbsto agglutinate bacteria of strain US46.

c In these ELISAs, the purified fimbriae of strain US46 or US5 were coatedon the solid phase. These titers were the highest dilution at which the opticaldensity at 490 nm was more than 0.2.

ELISA were similar for these three MAbs. No MAbsreacted with the fimbriae of strain US5 in the ELISA (Table1). In immunoblot analysis, all MAbs reacted with thepeptide band with the molecular weight of 20,000 and faintlywith the band of 41,000 molecular weight, but not with anyother band (Fig. 2). Since MAb DH2 showed a higheragglutination titer than the other IgG-class MAb (DC3), DH2was chosen for further experiments. To confirm the speci-ficity that DH2 had for the fimbriae, whole bacteria of strainUS46 or US5 were incubated with DH2 and labeled withprotein A-colloidal gold for electron microscopy. Gold par-ticles were observed only on the fimbriae and not on thebacterial surfaces of US46, and no particle attached to thefimbriae of US5 (Fig. 3). To identify the morphological typeof fimbriae recognized by DH2, we incubated the purifiedfimbriae of strain US46 with DH2. After washing, theagglutinate was further incubated with colloidal gold-conjugated secondary antibodies and was examined by elec-tron microscopy. The agglutinate was composed of only onetype of fimbriae (the 7-nm-wide type), and gold particles

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FIG. 4. Immunoelectron micrographs of negatively stained puri-fied fimbriae from strain US46. Purified fimbriae agglutinated withMAb DH2 were washed extensively to remove the other unreactedtypes of fimbriae and then were incubated with colloidal gold-conjugated secondary antibodies. These clusters of fimbriae werenegatively stained for electron microscopic examination. Only onetype of fimbriae, with a width of 7 nm, was observed, and goldparticles were attached to these fimbriae.

were observed around this type of fimbria (Fig. 4). Wefurther examined the mannose sensitivity of the fimbrial typethat reacted with MAb DH2. Chicken RBCs were incubatedwith purified fimbriae from strain US46 or US5 in thepresence of mannose to permit MR fimbriae to attach to the

B

0.5 p-m 0'1'.5 FIMFIG. 3. Immunoelectron micrographs of strains US46 (A) and US5 (B). The bacteria were incubated with MAb DH2 followed by

incubation with protein A-colloidal gold and were negatively stained for electron microscopic examination. The gold particles may be seenon the fimbriae of strain US46 but not on those of strain US5.

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FIG. 5. Immunoelectron micrographs of chicken RBCs. Chicken RBCs were incubated with purified fimbriae in the presence of mannose.After being washed, the RBCs were incubated with MAb DH2 and labeled with protein A-colloidal gold. Gold particles were attached to theerythrocytes incubated with US46 fimbriae (A), but no particles were attached to those incubated with US5 fimbriae (B).

RBCs. The reactivity of DH2 to the fimbriae on RBCs wasexamined with a colloidal gold probe. Gold particles wereobserved on the surfaces of RBCs incubated with US46fimbriae, but not on those incubated with US5 fimbriae (Fig.5).

Antigenic cross-reactivity of S. marcescens fimbriae. Theantigenic cross-reactivity of the fimbriae from several strainswas tested by MAb-mediated bacterial agglutination. A totalof 149 clinical isolates of S. marcescens were collected fromtwo different hospitals. Seventy strains (47% of the isolatedstrains) had hemagglutinating activity (HA) for guinea pig orchicken RBCs, or for both. No strain agglutinated sheep orhuman type A RBCs. The number of strains having MRHAor MSHA was 32 and 38, respectively. All MR strains wereagglutinated by MAb DH2 (Table 2). This result was furtherconfirmed by the spot test, using crude fimbriae of severalMR strains of S. marcescens as the antigen (Fig. 6). DH2reacted with crude fimbriae of randomly selected MR strainsbut not with the fimbriae of strain US5. Some strains havingMSHA were also agglutinated by DH2 (Table 2). These MSstrains were also agglutinated by polyclonal sera against US5fimbriae (data not shown). The US46 MR fimbriae, recog-nized by MAb DH2, were serologically different from theMS fimbriae of strain US5, as described above. Therefore, it

TABLE 2. Antigenic cross-reactivity of S. marcescens fimbriaeexamined with the MAb against the MR fimbriae of strain US46

No. of MR strainsa No. of MS strains"Source of agglutinated with agglutinated withstrains DH2 (%) DH2 (%)

Urine 14/14 (100) 6/17 (35)Sputum 18/18 (100) 2/21 (10)

Total 32/32 (100) 8/38 (21)a All tested strains showed hemagglutination of chicken RBCs. The per-

centage of agglutination-positive strains is shown in parentheses.

appears that such MS strains, agglutinated by DH2, have atleast two serologically different fimbriae.

Cross-reactivity of DH2 with other bacterial species. Vari-ous species including E. coli, Klebsiella spp., and Entero-bacter spp. were collected from clinical sources at Univer-sity Hospitals, and hemagglutinating strains were selected.All the tested strains showed either MRHA or MSHA forchicken, guinea pig, or human type A RBCs. No clinicalisolates of E. coli and Enterobacter spp. were agglutinatedby DH2 (Table 3). This MAb did not react with the well-known E. coli fimbriae CFAI, CFAII, F7, F8, F9, or Fil. Incontrast to these strains, many strains of Klebsiella spp.were agglutinated by DH2 (Table 3).

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L FFIG. 6. Spot test to determine the reactivity of MAb DH2 with

the fimbriae of several S. marcescens MR strains and strain US5.(A) Strain US5; (B) US14; (C) US17; (D) US24; (E) US30; (F) US54;(G) US46; (H) SS29; (1) SS32; (J) SS50; (K) SS60; and (L) SS67.

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TABLE 3. Interspecies antigenic cross-reactivity of fimbriaeexamined with DH2 against the fimbriae of S. marcescens

Species Hemagglutinating Strains agglutinatedstrainsa with DH2 (%)b

E. coli 17/44a 0/17 (0)Klebsiella spp. 21/60 7/21 (33)Enterobacter spp. 8/28 0/8 (0)

a Number of strains having HA/total number of collected strains.b Number of strains agglutinated with DH2/number of strains having HA.

DISCUSSION

Strain US46 had three different types of fimbriae whichcould be differentiated morphologically by electron micros-copy and chemically by SDS-PAGE. The widths of thefimbriae were 7, 5, and 3 nm, respectively, and the molecularweights of the subunit proteins were 21,000, 20,000, and19,000. We obtained a series of MAbs reacting with thefimbriae of US46, but not with those of US5, which has onlyMS fimbriae (15). The MAb DH2, chosen as a representativeof the MAbs, reacted with 7-nm-thick MR fimbriae and withthe 20,000-dalton peptide band. We examined the serologicalhomogeneity of fimbriae from clinically isolated strains of S.marcescens by the MAb-mediated bacterial agglutinationtest. We found that all strains having MRHA were aggluti-nated by MAb DH2. This result suggests that the antigenicdeterminant recognized by DH2 is widely distributed on theMR fimbriae of S. marcescens strains. A few strains showingMS hemagglutination also reacted with the MAb. Thesestrains were also agglutinated with polyclonal antibodiesagainst US5 fimbriae. DH2 did not react with US5 fimbriaein an ELISA, an immunoelectron microscopic study, or aspot test. The MR fimbriae of strain US46 and the MSfimbriae of strain US5 were serologically different. There-fore, these MS strains are proposed to have at least twodifferent types of fimbriae, one which reacts with DH2 andanother which is antigenically cross-reactive with the US5fimbriae. It seemed that many S. marcescens strains weremultifimbriated, and the mannose sensitivity of each strainwas dependent on the proportion of MR fimbriae to MSfimbriae.Most S. marcescens strains are reported to be multifimbri-

ated (1), as are strains of E. coli (3, 7). Because of thischaracter of fimbriated organisms, it has not been possible toperform a serological examination of fimbriae with polyclo-nal antisera. All our strains of S. marcescens which had HAwere agglutinated by yeast cells (data not shown), and someMS strains were agglutinated by MAbs against US46 MRfimbriae. It appeared that all the HA-positive strains had MShemagglutinins and that some MS strains had MR fimbriae.Therefore, many of the strains used in our study were alsomultifimbriated.Our study showed that the MR fimbriae were relatively

homogeneous, in terms of serological properties. Rothbardet al. (30) reported that the BrCN-cleaved peptide of thegonococcal pilin molecule contained an immunorecessiveepitope common to all gonococcal pilins which were sero-logically variable. The results of bacterial agglutination testswith polyclonal antibodies were exactly the same as thoseobtained with MAbs (data not shown). It appears that theepitope recognized by the MAbs used in our study is notimmunorecessive. Moreover, we obtained the MAb againstthe MR fimbriae of another S. marcescens strain, and thepreliminary experiments showed a similar result concerningthe antigenic cross-reactivity of S. marcescens MR fimbriae

by MAb-mediated bacterial agglutination tests (data notshown).Zak et al. (35) reported the expression of antigenically

distinct fimbriae in variants of an N. gonorrhoeae strainisolated from different anatomic locations and indicated animportant role for the widespread antigenic shift in thepathogenesis of N. gonorrhoeae. We examined MR strainsof S. marcescens derived from the urinary and respiratorytracts. The MR strains derived from both sites reactedequally with MAbs against the fimbriae of strain US46.Unlike N. gonorrheae, the S. marcescens strains derivedfrom different anatomic sites apparently do not expressserologically different fimbriae.The antigenic determinant of fimbriae has been exten-

sively studied in the fimbriae of N. gonorrhoeae. Rothbardet al. (30) reported that the disulfide loop peptide near thecarboxyl terminus of the subunit protein is an immuno-dominant region of this subunit and that this region isremarkably variable. The genetic analysis of gonococcalfimbriae revealed that multiple loci encoding various anti-genic determinants are localized on the chromosome (11). InE. coli, there are numerous serologically different fimbriaeon strains isolated from diverse origins. These fimbriaeseemed to be encoded by different genes, some of which arecarried by plasmids (8, 24). In contrast to these bacteria, theserological homogeneity of the MR fimbriae of S. marces-cens suggests that the gene encoding the antigenic determi-nant of MR fimbriae is highly conserved.Many Klebsiella strains having HA were agglutinated with

MAbs against fimbriae of S. marcescens. This result sug-gests that some Klebsiella strains express fimbriae serolog-ically identical to those of S. marcescens. Such interspeciescross-reaction of fimbriae was reported by Duguid et al. (4)in strains of Salmonella flexneri and E. coli. It is interestingthat Ottow (27) classified the fimbriae of Klebsiella spp. andS. marcescens into the same group, as determined from themorphological properties and the HA.

In other species, the serological heterogeneity of fimbriaeis generally high (10, 14, 22, 25, 26, 28, 33). To raise effectivevaccines against such bacteria, a mixture of a large numberof antigenically distinct fimbriae may be required. On thecontrary, the present study with MAbs revealed that theserological homogeneity of S. marcescens fimbriae was highcompared with that of fimbriae in gram-negative rods. There-fore, it seems that an effective vaccine must be composed ofonly a few types of fimbriae to prevent infections caused byS. marcescens.

ACKNOWLEDGMENTS

We thank A. Takade for assistance with the electron microscopicstudies, E. Yokota, K. Takemori, and K. Ohkochi for help incollecting the clinical isolates, and M. Ohara for comments on themanuscripts.

LITERATURE CITED1. Adegbola, R. A., and D. C. Olds. 1982. New fimbrial hemagglu-

tination in Serratia species. Infect. Immun. 38:306-315.2. de Ree, J. M., P. Schwillens, and J. F. van den Bosch. 1985.

Monoclonal antibodies that recognize the P fimbriae F71, F72,F9, and Fll from uropathogenic Escherichia coli. Infect. Im-mun. 50:900-904.

3. Duguid, J. P., S. Clegg, and M. I. Wilson. 1979. The fimbrial andnon-fimbrial haemagglutinins of Escherichia coli. J. Med. Mi-crobiol. 12:213-227.

4. Duguid, J. P., and R. R. Gillies. 1958. Fimbriae and hemagglu-tinating activity in Salmonella, Klebsiella, Proteus and Chro-mobacterium. J. Pathol. Bacteriol. 75:519-520.

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