JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1988, p. 2402-2409 Vol. 26, No. 110095-1137/88/112402-08$02.00/0Copyright (O 1988, American Society for Microbiology

Phenotypic and Genotypic Characterization of EnterotoxigenicEscherichia coli Serotype 08:KX105 and 08:K"2829" Strains

Isolated from Piglets with DiarrheaANDRE BROES,1* JOHN M. FAIRBROTHER,' JACQUES MAINIL,2 JOSEE HAREL,1 AND SERGE LARIVIERE'Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe,

Quebec, Canada J2S 7C6,' and Chaire de Bactériologie et de Pathologie des Maladies Bactériennes, Faculté deMédecine Vétérinaire, Université de Liège, B-1070 Brussels, Belgium2

Received 4 May 1988/Accepted 12 July 1988

Twelve pathogenic and seven nonpathogenic enterotoxigenic Escherichia coli strains which were previouslyidentified as belonging to serogroup 08:KX105 (A. Broes, J. M. Fairbrother, S. Larivière, M. Jacques, andW. M. Johnson, Infect. Immun. 56:241-246, 1988) were further examined for their phenotypic and genotypicproperties. Only the 12 pathogenic strains were confirmed to possess the capsular antigen KX105. The sevennonpathogenic strains did not possess this antigen and thus were incorrectly assigned to have capsular antigenKX105. All seven nonpathogenic strains apparently possessed a previously unrecognized capsular antigenwhich has been designated K"2829". Studies with antisera prepared against FI (type 1) fimbriae from threeE. coli strains suggested that at least three antigenic subtypes of FI fimbriae were represented among the 08:KX105 strains examined. By using serotyping, biotyping, and outer membrane protein profile analyses, the 08:KX105 strains were divided into at least two distinct clusters, whereas the 08:K"2829" strains were groupedinto a single unique cluster. Most of the strains of the same cluster were further differentiated by testing forantibiotic resistance and colicin production and resistance and by analysis of plasmid content. With theexception of one strain which lost its enterotoxicity during storage, all of the 08:KX105 strains hybridized withthe gene probes for the heat-labile (LT) and heat-stable (STb) enterotoxins. For each 08:KX105 strain, a singleplasmid ranging in size from 61 to 77 megadaltons carried the LT and STb genes. All of the enterotoxigenic 08:KX105 strains fermented sorbose, whereas the nonenterotoxigenic strain did not. All of the 08:K"2829"strains hybridized with the STb probe only. For each 08:K"2829" strain, the STb genes were located on asingle plasmid of 61 or 22 megadaltons. None of the strains demonstrated homology with the genes encodingthe F4 (K88), F5 (K99), F6 (987P), and F41 fimbrial antigens and STaP and STaH.

Enterotoxigenic Escherichia coli (ETEC) strains are animportant cause of diarrhea in piglets (26). Most ETECstrains belong to a limited number of O and K serogroups,produce heat-labile enterotoxin (LT), heat-stable enterotox-ins (STa and STb), or both. These enterotoxins causeintestinal fluid secretion, resulting in watery diarrhea, andexpress fimbrial adhesins F4 (K88), F5 (K99), F6 (987P), orF41 (see reference 31 for a discussion of antigen nomencla-ture), which allow the organisms to colonize the smallintestine by adhering to the mucosa (26). The genes codingfor the enterotoxins LT (39), STa (40), and STb (17), as wellas those coding for the fimbriae F4 (24) and F5 (43), arelocated on plasmids, whereas those coding for the adhesinsF6 (6, 7, 27) and F41 (28) are located on the chromosome.These genes have been cloned and can be detected by DNAhybridization techniques by using radiolabeled gene probesderived from recombinant plasmids (20, 21, 25).Although the different O and K antigens, enterotoxins,

and fimbrial adhesins are usually not genetically linked inporcine ETEC strains, a close association between expres-sion of certain O and K serogroups, enterotoxins, andfimbrial adhesins has been noted (26). Relationships betweenEscherichia coli strains can be investigated by using severalprocedures, including 0, K, and H serotyping; hemaggluti-nation pattern; toxin production; antibiotic susceptibility;metabolic properties; plasmid content; restriction endonu-clease DNA analysis; and lipopolysaccharide, outer mem-

* Corresponding author.

brane protein (OMP), and enzyme electrophoretic profiles(2, 5, 12, 30, 31, 34). Such characteristics have provided thebasis for the concept of the clonal structure of pathogenic E.coli populations (31, 34).ETEC 08:KX105 strains have been associated with diar-

rhea in piglets in The Netherlands and the province ofQuebec (3). In a previous study (3), we reported that (i) onlycertain strains identified as belonging to this serogroup causediarrhea in orally infected, colostrum-deprived newbornpiglets; (ii) the pathogenic strains produced both LT andSTb; (iii) the nonpathogenic strains produced STb only; and(iv) the pathogenic strains were negative for the F4, F5, andF41 fimbrial adhesins but that most of them reacted with F6antiserum. However, this last reaction was observed in theintestines of experimentally infected piglets but not in cul-ture. Thus, the purpose of the present study was to furthercharacterize these strains, in order to elucidate relationshipsbetween pathogenic and nonpathogenic strains and to verifythe presence of the gene encoding the F6 fimbrial adhesin inthe pathogenic strains.

MATERIALS AND METHODS

Bacterial strains. The 19 ETEC 08 strains examined in thisstudy were previously characterized for their pathogenicitiesin piglets and for the production of virulence factors (3).Twelve of these strains were pathogenic for colostrum-deprived newborn piglets. In addition, the porcine ETECstrain 3539 (0115:H-; J. M. Fairbrother, Université deMontréal, Montreal, Quebec, Canada); a human pyeloneph-

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CHARACTERIZATION OF ETEC STRAINS 2403

ritic E. coli strain, KS71 (04:K12:H-) (T. K. Korhonen,University of Helsinki, Helsinki, Finland); and a Fl (type 1)fimbriae prototype laboratory E. coli strain, BAM (rough),were used for the production of antisera against Fl fimbriae.Strains K-12(pRIT10036) (STaP+), K-12(pRIT10250)(STaH+), K-12(pRAS1) (STb+), K-12(pEWD299) (LT+),K-12(pMKO05) (F4+), K-12(pFK99) (F5+), K-12(pPK150)(F6+), HB101 (K-12), P307 (STb+ LT+), H10407 (STaP+STaH+ LT+), Moon-1790 (K-12- STb+), 82-1128 (F4+STb+ LT+), B44 (STa+ F5' F41+), Moon-431 (STa+ F5+),Moon-987 (STa+ STb+ F6+), Moon-1592 (STa+ STb+ F6+),603A (STa+ F6+), HB101(pPK150) (F6+), and 4787 (F165+)were used for the preparation of gene probes or as controls.O, K, and H serotyping. 0, K, and H serotyping was

performed at the International Escherichia and KlebsiellaCentre (Copenhagen, Denmark) as described previously(30).Fl serotyping. Strains were examined for Fl fimbrial

antigens by using the indirect fluorescent antibody test withrabbit antisera raised against Fl fimbriae purified fromstrains 3539, KS71, and BAM. Purification of Fl fimbriaefrom strains 3539 and BAM was carried out as describedpreviously (3). A single 17-kilodalton (kDa) peptide bandwas observed by sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE) of both fimbrialpreparations after they were treated with acid (HCl; pH 1.8)as described previously (23). Immunization of rabbits withpurified fimbriae was carried out as described previously (3).Rabbit antiserum against Fl fimbriae (KS71D) from strainKS71 was kindly provided by P. Lintermans (Institut Na-tional de Recherches Vétérinaires, Brussels, Belgium). Forthe indirect fluorescent antibody test, bacteria were grownovernight at 37°C in tryptic soy broth (Difco Laboratories,Detroit, Mich.), recovered by centrifugation (8,000 x g for20 min), washed in phosphate-buffered saline, and sus-pended to a standard density of about 109 cells per ml.Smears made from bacterial suspensions onto glass slideswere air dried and heat fixed. They were incubated with theappropriate anti-Fl antisera (dilution, 1:50) for 30 min in amoist chamber at 37°C, rinsed in phosphate-buffered salineand distilled water, and then incubated in the appropriatedilution of fluorescein-conjugated goat anti-rabbit immuno-globulin G (Bio/Can Scientific Inc., Mississauga, Ontario,Canada) in a moist chamber at 37°C for 30 min. Slides wereexamined with a fluorescence microscope (Laborlux 12) withepi-illumunation (Leitz, Wetzlar, Federal Republic of Ger-many). The intensity of the bacterial fluorescence stainingwas recorded by using an index of0 (no fluorescence) to 4(intense fluorescence).

Detection of hemolysin. Strains were examined for hemo-lysin production after overnight incubation at 37°C on bloodagar base (Difco)-5% calf blood (blood agar).

Biotyping. Biotyping was performed by the procedure ofCrichton and Old (5). Amino acid decarboxylation tests wereconducted in Moeller decarboxylation base (Difco) mediumwith 0.5% (wt/vol) L-lysine or L-ornithine. Sterile mineral oilwas added to each tube after inoculation. Results wererecorded after incubation at 37°C for 48 h. Carbohydratefermentation tests were performed in purple broth base(Difco) medium with 0.5% (wt/vol) L-sorbose, raffinose,dulcitol, or L-rhamnose. Tubes were incubated at 37°C for 24h for those containing L-rhamnose and L-sorbose, 48 h forthose containing dulcitol, and 72 h for those containingraffinose. Hydrolysis of esculin was assessed on nutrientagar-0.1% (wt/vol) esculin-0.05% (wt/vol) ferric citrate.Results were recorded after incubation at 37°C for 72 h.

Motility was evaluated by phase-contrast microscopic exam-ination of bacteria grown overnight at 37°C in tryptic soybroth. Production of Fl fimbriae was determined by man-nose-sensitive agglutination of guinea pig erythrocytes andnegative staining transmission electron microscopy as de-scribed previously (3). The ability to grow on minimalmedium was determined by culturing strains onto glucose(0.3% [wt/vol]) mineral salt agar (5).

Antibiotic susceptibility. Susceptibility to ampicillin, ceph-alothin, chloramphenicol, gentamicin, kanamycin, specti-nomycin, streptomycin, tetracycline, sulfonamides, and tri-methoprim-sulfamethoxazole was determined by the agardisk diffusion technique by the procedure of Kirby-Bauer(1).

Colicin production. Production of colicins was determinedby the agar overlay technique, as described previously (11).Briefly, strains were stabbed onto blood agar plates andincubated for 48 h. The cells were then killed by exposure tochloroform vapor for 30 min, and the plates were overlaidwith 5 ml of tryptic soy broth with 0.75% agar containingapproximately 5 x 107 cells of the colicin indicator E. coliK-12 ROW. Inhibition zones were examined after overnightincubation at 37°C.

Colicin susceptibility. Strains were examined for suscepti-bility to the colicins produced by the type strains CA31(colicin A), CA18 (colicin B), TAM582/T5 (colicin B+M),CA23 (colicin D), CA38 (colicin E+I), CA42 (colicin F),CA53 (colicin la), MR2 (colicin lb), CA62 (colicin J+I),K235 (colicin K and undetermined colicin), K49 (colicin K),K302 (colicin S5), and I.7a (colicin V) by the agar overlaymethod (11). These strains originated from P. Frédéricq(Institut de Microbiologie et d'Hygiène, University of Liège,Liege, Belgium) and were kindly provided by P. Pohl(Institut National de Recherches Vétérinaires, Brussels,Belgium).

Preparation of membrane proteins. Bacterial membraneswere prepared by a modification of the method of Lugten-berg et al. (18). Briefly, bacteria were grown with agitation intryptic soy broth at 37°C overnight. Cells were collected bycentrifugation and suspended in 50 mM Tris hydrochloride(pH 7.8)-l mM EDTA. The suspension was vortexed vigor-ously for 2 min and was then centrifuged at 4,500 x g for 15min. The cell pellet was suspended in 50 mM Tris hydro-chloride (pH 7.8)-l mM EDTA and sonicated 4 times for 15s each time by using an ultrasonic disintegrator (Vibra Cell;Sonics & Materials Inc., Danbury, Conn.). The resultingsuspension was centrifuged at 1,200 x g for 20 min, thepellet was discarded, and the supernatant was centrifuged at100,000 x g at 40C for 1 h. The pellet representing a crudebacterial membrane preparation was examined by SDS-PAGE.SDS-PAGE. Discontinuous SDS-PAGE was performed by

a modification of the procedure described by Laemmli (16).Stacking and separating acrylamide gels of 4.0% (0.125 MTris hydrochloride [pH 6.8]) and 10% (0.375 M Tris hydro-chloride [pH 8.8]), respectively, were prepared in a mini-gelapparatus (Mini-Protean II system; Bio-Rad Laboratories,Richmond, Calif.). Electrophoresis buffer was 25 mM Trishydrochloride-0.2 M glycine solution (pH 8.3) with 0.1%SDS. Samples were diluted 1:1 in 60 mM Tris hydrochloridebuffer with 2% SDS-10% glycerol-0.002% bromophenolblue-5% P3-mercaptoethanol and boiled for 5 min. Electro-phoresis was carried out at a constant voltage of 200 V forabout 45 min. A low-molecular-weight electrophoresis kit(Bio-Rad) was used as a standard. Gels were stained with

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2404 BROES ET AL.

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I kb 39 81 128,l16.5120.61FIG. 1. Linear map of plasmid pPK150 according to de Graaf and Klaasen (6). Restriction sites for restriction endonucleases BamHI (Ba),

HindIII (Hi), BglII (Bg), HpaI (Hp), and Sphl (Sp) are shown. Numbers in the open bars refer to sizes (in kilodaltons) of polypeptidesinvolved in the biosynthesis of F6 (987P) fimbriae (the F6 structural subunit is the 20.5-kDa polypeptide). Probes A and B are indicated. kb,Kilobases.

Coomassie brilliant blue R250 and were destained in asolution of 40% methanol-70% acetic acid.

Plasmid DNA isolation. Plasmid DNA was extracted fromcells by an adaptation of the method of Kado and Liu (15).Briefly, bacteria were grown in L broth (containing thefollowing per liter: 10 g of peptone, 5 g of yeast extract, 10 gof NaCi, and 1 g of glucose [pH 7.2]) overnight at 37°C, and1.5 ml of the culture was centrifuged in a microfuge (Eppen-dorf) for S min. The cell pellet was suspended in 200 ,ul of 10mM Tris hydrochloride (pH 8.0)-i mM EDTA buffer, mixedwith 400 ptI of freshly prepared lysing solution (50 mM Trisand 3% SDS; pH adjusted to 12.6 with 10 N NaOH), andincubated at 65°C for 1 h. An equal volume of phenol-chloroform (1:1 [vol/vol]) was then added, and the suspen-sion was emulsified by mixing and then centrifuged for 15min. The upper aqueous phase was carefully removed andkept at 4°C until analysis. Three extractions were performedfor each strain, and the extracts were pooled.DNA gel electrophoresis. DNA was electrophoresed in a

0.5% agarose gel in 40 mM Tris acetate-2 mM EDTA (pH7.9) buffer (22) at 2.5 V/cm overnight at 4°C. Gels werestained with ethidium bromide (1 ,ug/ml), rinsed, and thenphotographed under a shortwave UV light source with anorange filter. The molecular weight of each plasmid wasestimated by comparison with plasmids of Erwinia uredo-vora 391 (42).

Preparation of DNA probes. Gene probes for the entero-toxins STaP, STaH, STb, and LT and the fimbriae F4 and F5were derived from recombinant E. coli K-12 strains contain-ing the plasmids pRIT10036, pRIT10250, pRAS1, pEWD299,pMKO05, and pFK99 respectively, as described previously(20, 21, 28). Two different gene probes for the F6 fimbriaewere derived from the recombinant strain E. coli K-12(pPK150) (kindly provided by F. K. De Graaf, VrijeUniversiteit, Amsterdam, The Netherlands) (6, 7). The firstprobe (probe A) was the 1.3-kilobase fragment generated bydigestion with HpaI and BglII, and the second probe (probeB) was the 3-kilobase fragment generated by digestion withHpaI and SphI (Fig. 1). Plasmid DNA was extracted andpurified by ultracentrifugation in a cesium chloride gradient(22). Plasmids were digested with the appropriate restrictionendonucleases under conditions specified by the manufac-turer (Boehringer GmbH, Mannheim, Federal Republic ofGermany). The resulting fragments were separated by aga-

rose gel electrophoresis or PAGE. Appropriate fragmentswere cut from the gel, recovered by electroelution, and

concentrated by ethanol precipitation. DNA fragments werelabeled with [t-32P]dCTP by using a multiprimer DNAlabeling kit (Amersham Corp., Arlington Heights, 111.) ac-cording to the instructions of the manufacturer.Colony hybridizations. Strains were spot inoculated onto L

agar (L broth containing 15 g of agar per liter) and incubatedat 37°C for 18 h. Colonies were replicated by placing filterpaper (541; Whatman, Inc., Clifton, N.J.) on the surface ofthe agar for 2 h. The paper filters were then peeled off andconsecutively placed colony side up onto Whatman 3MMfilter papers saturated with the following solutions: (i) 10%SDS for 3 min, (ii) 0.5 M NaOH-1.5 M NaCl for 15 min, and(iii) 0.5 M Tris hydrochloride (pH 7.5)-1.5 M NaCI twice for15 min each time. Filters were then air dried and stored atroom temperature. For the hybridization, filters were placedin hybridization buffer which consisted of 3 x SSC (lx SSCis 0.15 M NaCI plus 0.015 M sodium citrate), 10x Denhardtsolution (lx Denhardt solution is 0.02% Ficoll [molecularweight, 400,000; Pharmacia Fine Chemicals, Piscataway,N.J.], 0.02% polyvinylpyrrolidone [molecular weight,360,000], and 0.02% bovine serum albumin), 1% heat-dena-tured salmon sperm DNA, and 0.1% SDS (22) for 15 min at65°C and then transferred to fresh hybridization solutioncontaining about S x 105 cpm of appropriate heat-denatured,32P-labeled DNA probe per filter. After hybridization underagitation at 65°C overnight, the filters were washed in 3xSSC with 0.1% SDS 3 times for 30 min each time at 65°C andair dried. The filters were air dried and exposed to X-ray film(X-Omat-R; Eastman Kodak Co., Rochester, N.Y.) for 1 to2 days at -70°C. The film was developed according to theinstructions of the manufacturer. Strains HB101, H10407,Moon-1790, 82-1128, B44, Moon-431, Moon-987, Moon-1592, 603A, HB101(pPK150), and 4787 were used as con-trols.

Hybridizations in agarose gels. Plasmids separated byagarose gel electrophoresis were hybridized with the STband LT gene probes by a modification of the techniquedescribed by Maas et al. (19; J. Mainil, F. Bex, P. Dreze, A.Kaeckenbeeck, and M. Couturier, manuscript in prepara-tion). After electrophoresis, gels were denatured in 0.5 MNaOH-0.6 M NaCl for 30 min at 37°C with agitation and thenwashed with 0.1 M Tris hydrochloride (pH 7.5)-1.5 M NaClat room temperature for 15 min. They were then mounted insandwich form between two pieces of Whatman 3MM filterpaper and dried by using a gel dryer (Bio-Rad) operating witha vacuum but with no heat for about 15 min. Dried gels were

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CHARACTERIZATION OF ETEC STRAINS 2405

TABLE 1. Characteristics of ETEC 08:KX105 and 08:K"2829" strains isolated from piglets with diarrhea

Strain K and H gru Biotypea Resistance to antibioticsb (2) OMP Production Susceptibility to DNA probe Plasmid content'antigens group profile of colicins colicins hybridization

85-3033 KX1O5:H- 1 lde A, Cm, N, K, Sp, S, T, Su A - F, X LT+ STb+ 105, 72, 579-1541 KX1O5:H9 4df A, N, K, S, T, Su B V+? F, X LT+ STb+ 73, 69, 67, 4080-1385 KX1O5:H9 4df A, N, K, S, T, Su B V+? F, X LT+ STb+ 73, 69, 67, 4085-1134 KX1O5:H9 2 4d A, Cm, N, K, Sp, S, T, Su B - F, X LT+ STb+ 73, 69, 67, 586-0484 KX1O5:H9 2 4d A, Cm,N, K, Sp, S, T, Su B - F, X LT+ STb+ 73, 69, 67, 5085-1233 KX1O5:H9 2 4d A, Cm, N, K, Sp, S, T, Su B - A, F, I, K, X LT+ STb+ 77, 67, 64, 2181-2319 KX1O5:H- 1 4dep Sp, S, T, Su B - F, X LT+ STb+ 69, 64, 2584-4403 KX1O5:H9 3 6d A, Cm, N, K, Sp, S, T, Su B - X 73, 4380-7169 KX1O5:H14 lldf Ce, Sp, S, T, Su A - B, D, E, F, J, M, X LT+ STb+ 67, 62, 48, 30, 584-4414 KX1O5:H14 3 llcd S, T, Su A - F, M, X LT+ STb+ 64, 586-1061 KX1O5:H14 3 llcd N, K, S, T, Su A - D, F, M, X LT+ STb+ 67, 52, 5Guinée KX1O5:H14 3 12d S, T A - F, M, X LT+ STb+ 67, 64, 61, 35

82-2829 K"2829":H- 16(d)ef S, T C - E, F, J, M, X STb+ 61, 55, 4781-2714 K"2829":H- 16(d)ef Cm, N, K, Sp, S, T, Su C - E, F, J, M, X STb+ 68, 61, 55, 47, 584-0569 K"2829":H 16(d)ef Cm, N, K, Sp, S, T, Su C - F. M, X STb+ 68, 61, 4784-3225 K"2829":H- 16(d)ef A, Sp, S, T, Su C - F, t, M, X STb+ 61, 52, 3286-1276 K"2829":H- 2 16cde A, Sp, S, T, Su C - F, M, X STb+ 61, 52, 2282-0817 K"2829":H- 16cdef A, Ce, N, K, Sp, S, T, Su C - F, M, X STb+ 52, 47, 2283-3645 K"2829":H- 16cdef A, N, K, Sp, S, T, Su C - F, M, X STb+ 52, 47, 22

a Biotypes are as described by Crichton and Old (5): b, negative for rhamnose; c, negative for lysine; d, negative for esculin; (d), both esculin-positive and-negative colonies; e, nonmotile; f, negative for FI fimbriae; p. no growth on minimal glucose medium.

b Abbreviations: A, ampicllin; Ce, cephalothin; Cm, chloramphenicol; G, gentamicin; K, kanamycin; Sp, spectinomycin; S, streptomycin; T, tetracycline; Su,sulfonamides; T, Su, trimethoprim-sulfamethoxazole.

C The size of plasmid is expressed in megadaltons; the plasmid hybridizing with the LT probe, the STb probe, or both probes in boldface.

then immersed in a 0.1 M Tris hydrochloride (pH 7.5)-1.5 MNaCI solution for about 15 min to allow detachment from theWhatman 3MM filter paper. Gels were prehybridized for 1 hat 65°C in hybridization solution and then hybridized asdescribed above. About 106 cpm of the appropriately heat-denatured, 32P-labeled DNA probe was used per gel. Afterfinal washings, gels were air dried and autoradiographed at-70°C for 2 to 7 days. Plasmid P307 containing the LT andSTb genes was used as the positive control (17, 39).

RESULTS

K serotyping. Only the 12 pathogenic strains possessed thecapsular KX105 antigen (Table 1). In contrast, the sevennonpathogenic strains were found to be negative for thisantigen. Using the agglutination test with an antiserumprepared against the nonpathogenic strain 2829, we foundthat all of these strains apparently shared the same uniden-tified polysaccharide capsular antigen (K"2829").H serotyping. Ten of the 08:KX105 strains were motile

and possessed the flagellar antigens H9 or H14, whereas the08:K"2829" strains were nonmotile (Table 1).Fl serotyping. A total of 9 of the 12 08:KX105 strains and

1 of the 7 08:K"2829" strains produced Fl fimbriae, asdemonstrated by transmission electron microscopy andmannose-sensitive hemagglutination of guinea pig erythro-cytes. Based on their reaction in the indirect fluorescentantibody test with the three Fl antisera, these strains wereclassified into three distinct antigenical subtypes (Table 1).Two strains reacted strongly (4+ reaction) with all threeantisera (group 1), four strains reacted strongly (4+ reaction)with anti-3539 and anti-BAM antisera but only weakly (1+reaction) with anti-KS71D antiserum (group 2), and fourstrains reacted strongly (4+ reaction) with anti-KS71D andanti-BAM antisera but only weakly (1+ reaction) with anti-3539 antiserum (group 3).

Hemolysis. Only two of the strains tested (1233 and 1134;both were 08:KX105) were hemolytic. Interestingly, strain

85-1134 produced both hemolytic and nonhemolytic colo-nies. These two variants were both enterotoxigenic andpathogenic for newborn piglets (unpublished data).

Biotyping. The 08:KX105 strains were classified into fiveprimary biotypes (biotypes 1, 4, 6, 11, and 12), with 9 of the12 strains belonging to biotypes 4 and 11 (Table 1). Incontrast, all ofthe 08:K"2829" strains belonged to the saneprimary biotype (biotype 16). All of the 08:KX105 strains,except strain 4403, fermented sorbose, whereas the 08:K"2829" strains did not ferment this sugar. Further differ-entiation of the strains was achieved by using secondarybiotyping (Table 1). However, the esculin hydrolysis testsused for secondary biotyping were difficult to interpret, assome strains produced both esculin-positive and esculin-negative colonies.

Antibiotic resistance. Multiple antibiotic resistance wasdemonstrated for most of the strains (Table 1). All but 3strains were resistant to at least four antibiotics, and 11strains were resistant to six or more antibiotics. Resistanceto streptomycin, tetracycline, and the sulfonamides was themost common. All of the strains remained susceptible totrimethoprim-sulfamethoxazole and gentamicin. In contrastto the strains isolated in Quebec, strain Guinée, whichoriginated from The Netherlands, was resistant only tostreptomycin and tetracycline.OMP profile. Three distinct OMP profiles were identified

and arbitrarily designated A, B, and C. Profile A wascharacterized by a single major 38-kDa polypeptide band,profile B by major polypeptide bands of 43.5 and 35.5 kDa,and profile C by major polypeptide bands of 38 and 35.5 kDa(Fig. 2). The 08:KX105 strains of biotypes 1, 11, and 12belonged to profile A, whereas strains of biotypes 4 and 6belonged to profile B (Table 1). In contrast, all of the 08:K"2829" strains belonged exclusively to profile C.

Colicin production and susceptibility. With the exception oftwo 08:KX105 strains which produced colicin V and anundetermined colicin, none of the other strains were colicin-

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1 2 3 4

FIG. 2. SDS-PAGE patterns of E. coli 08:KX105 and 08:K"2829" membrane extracts. Electrophoresis was conducted fromthe top (cathode) to the bottom (anode) in 10% acrylamide gels.Lanes 1, 2, and 3, Patterns C, B, and A, respectively; lane 4, themolecular weight standards (Bio-Rad) phosphorylase b (92,500),bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhy-drase (31,000), soybean trypsin inhibitor (21,500), and lysozyme(14,400), from top to bottom, respectively.

ogenic (Table 1). Most of the strains were susceptible to only2 or 3 of the 13 colicins tested (Table 1). All of the strainswere susceptible to colicins F and X, except for strain 4403,which was susceptible to colicin X only.

Plasmid content. All of the strains contained from two tofive plasmids ranging from about 5 to 106 MDa (Table 1).However, most were large plasmids of 40 to 80 MDa. Mostof the strains had unique plasmid profiles. However, the 08:KX105 strains 1541 and 1385 had identical plasmid profilesas did the 08:K"2829" strains 0817 and 3645. In addition,the 08:KX105 strains 1134 and 0484 had profiles whichdiffered by only one plasmid, as did the 08:K"2829" strains3225 and 1276. Strains with identical or similar plasmidprofiles were also very similar with respect to other pheno-typic properties.DNA hybridizations. None of the strains hybridized with

the gene probes for the F4 and F5 fimbrial antigens and theenterotoxins STaP and STaH. With the exception of onestrain (4403), all the 08:KX105 strains hybridized with theSTb and LT gene probes. Strain 4403, which did not hybrid-ize with either the STb or the LT probe, originally producedLT and STb and was the only sorbose-negative 08:KX105strain. For each of the 08:KX105 strains, only one plasmidhybridized with both the STb and LT probes, indicating thatgenes coding for these enterotoxins are located on a singleplasmid (Fig. 3). The plasmids containing STb and LT geneswere 64 MDa (two strains), 67 MDa (two strains), 69 MDa(five strains), 72 MDa (one strain), or 77 MDa (one strain)(Table 1). All of the 08:K"2829" strains hybridized with theSTb probe only. Only one plasmid hybridized with the STbprobe for each of the 08:K"2829" strains, and the STbplasmid was 61 MDa (five strains) or 22 MDa (two strains)(Table 1). When examined by colony hybridization understringent conditions, none of the strains demonstrated ho-mology with the gene coding for the F6 (987P) structuralsubunit. In contrast, the F6-positive control strains (Moon-987, Moon-1592, and 603A) and strain K-12(pPK150)strongly hybridized with the two probes that were used.

75 ô65- 9M _-" .

40..

c

75-65_@s__--

40-h ,FIG. 3. Agarose gel electrophoresis of plasmid DNA of 9 repre-

sentative 08:KX105 strains (A) and the corresponding autoradio-graphs of duplicate gels that show plasmids containing DNA se-quences which hybridized with the LT (B) and STb (C) probes. Foreach of these strains, only one plasmid hybridized with both the STband LT probes. Molecular sizes are in megadaltons.

DISCUSSIONWe reported previously that ETEC strains identified as

belonging to serogroup 08:KX105 included both pathogenicand nonpathogenic strains (3). In the present study, wedemonstrated that only the pathogenic strains possessed theKX105 capsular antigen and that the nonpathogenic strainswere misidentified previously. In the present study, K sero-typing was accomplished by use of counterimmunoelectro-phoresis, which is more reliable than the slide agglutinationtest used originally (3, 30). Interestingly, there was evidencethat all of the nonpathogenic strains shared the same capsu-lar antigen. These results thus suggest that there is anassociation between the presence of the capsular antigenKX105 and pathogenicity for piglets, and they demonstratethe importance of O and K serotyping as a diagnostic tool forthe identification of porcine ETEC strains (26, 30). Thepresence of certain true heat-stable (A-type) capsular anti-gens has been shown to enhance the ability of porcine andbovine ETEC strains to colonize the small intestine andincrease the virulence of these strains (38). Thus, it isimportant to examine the possible role of the KX105 capsule

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in the pathogenicity for piglets of the 08:KX105 strains (A.Broes, J. M. Fairbrother, M. Jacques, and S. Lariviere,Can. J. Vet. Res., in press).The combination of biotyping, serotyping, and OMP pro-

file analysis appeared to be a useful approach for studyingthe subpopulations of strains in the 08:KX105 and 08:K"2829" serogroups. These phenotypic characters are con-sidered stable and have been used to define bacterial clusters(2, 5, 29, 30, 34). Examination of these characters hasindicated that there is a structural heterogeneity among the08:KX105 strains, whereas the 08:K"2829" strains wererelatively homogeneous. These results suggest that the 08:K"2829" strains represent a single bacterial cluster,whereas the 08:KX105 strains are made up of at least twodistinct bacterial clusters, the first grouping strains ofbiotypes 4 and 6 and having antigen H9 and OMP profile B,and the second grouping strains of biotypes 1, 11, and 12 andhaving antigen H14 and OMP profile A. The determination ofthe electrophoretic types of the alloenzymes and DNAendonuclease restriction profiles, which is considered, alongwith OMP profiles, to be the best criteria for the definition ofbacterial clones, would be useful to test this hypothesis,although these techniques are cumbersome and are re-stricted to specialized laboratories (34). Further differentia-tion of strains of the same cluster was achieved by testing forsusceptibility to antibiotics and colicin production and sus-ceptibility and by analysis of plasmid content. Most of thestrains differed by at least one of these properties, althoughtwo pairs of strains (1541 and 1385, 1134 and 0484), whichwere isolated at different times from different swine herds,were indistinguishable with respect to these properties.An association between sorbose fermentation and LT

production was observed among the 08:KX105 strains(Table 1). Indeed, the only 08:KX105 strain which did notferment sorbose appeared to have lost its enterotoxicityduring storage. This observation suggests that the genescoding for the two properties are located on a single plasmid.In contrast, a negative correlation between enterotoxicityand sorbose fermentation has been noted among E. colistrains isolated from humans with diarrhea in Australia,New Zealand, and Indonesia (4). Linkage of genes control-ling production of the K88 fimbrial antigen and raffinosefermentation is a common feature in porcine ETEC strains(37). Similarly, an association between adonitol fermentationand production of the FS (K99) fimbrial antigen, which isusually associated with production of STa (37), has beenobserved in certain bovine ETEC strains (33).Most of the pathogenic 08:KX105 strains were found to

produce Fl fimbriae (3). In addition, these fimbriae areexpressed by bacteria in the intestines of newborn pigletsthat were experimentally infected with these strains (unpub-lished data). Fl fimbriae, which are characterized by theirability to bind to D-mannosides, are commonly expressed bymany E. coli strains (8, 31, 35). Several studies in whichpolyclonal antisera or monoclonal antibodies were used havedemonstrated an immunological heterogeneity among the Flfimbriae from E. coli (8, 13, 31, 35, 36, 41). Jayappa et al. (14)have suggested that Fl fimbriae from E. coli strains isolatedfrom humans consisted of at least 12 different serotypes,whereas those isolated from ETEC strains from pigs be-longed to only one serotype. In contrast, our own resultsprovide evidence that immunological heterogeneity alsoexists among Fl fimbriae from strains with the same 0:Kserogroup. Indeed, the Fl fimbriae from the 08:KX105strains examined in this study shared major common anti-genic determinant(s) expressed by prototype Fl strain BAM

but varied in the production of other specific determinant(s)exhibited by Fl strains 3539 and KS71. Although the role ofFl fimbriae in ETEC enteric infections is controversial, theincorporation of this antigen into vaccines for the control ofporcine ETEC diarrhea has been proposed (14). Our resultsunderline the need to further characterize the antigenic andfunctional properties of Fl fimbriae from porcine ETECstrains.We previously reported that the 08:KX105 and 08:

K"2829" strains neither produced STa nor expressed the F4(K88), F5 (K99), or F41 fimbrial antigens (3). Results ofhybridization tests with the STaP, STaH, F4 (K88), and F5(K99) gene probes confirmed those of previous phenotypictests (3). As the genes encoding the F4 (K88) and F41fimbrial antigens demonstrated extended homology, we canalso assume that the strains do not possess the genesencoding the F41 fimbrial antigen (28). Results of hybridiza-tion tests with the LT and STb gene probes were also inagreement with those of previous phenotypic tests (3).Except for one strain which lost its enterotoxicity duringstorage, all of the 08:KX105 strains hybridized with the LTand STb probes. All the strains carrying the LT genes alsocarried the STb genes, in each case on a single plasmid, thusconfirming previous observations of the association betweenLT and STb genes in porcine ETEC (9, 10, 37). Althoughplasmids containing LT and STb genes from porcine ETECstrains have not been studied extensively, there are indica-tions that these plasmids are closely related (37). The differ-ences observed in the size of the plasmids containing LT andSTb genes of the 08:KX105 strains did not exclude apossible relationship between them. These differences maybe due to the acquisition or loss of genetic elements such astransposons coding for antibiotic resistance. This hypothesisneeds to be examined further by means of incompatibilitystudies and replicon typing and by restriction endonucleaseanalysis.None of the strains demonstrated homology with the gene

coding for the F6 (987P) structural subunit. These resultsthus indicate that the 08:KX105 strains which were found toreact in the intestines of infected piglets with antiserumraised against purified F6 (987P) fimbriae (3) do not containthe gene coding for the F6 (987P) fimbrial subunit. Weformerly suspected that the 08:KX105 strains did not pro-duce typical F6 (987P) fimbrial adhesin because they did notcolonize the small intestine as intensively as classical F6-positive strains did and because they originated from weanedas well as suckling piglets, whereas classical F6-positivestrains were usually isolated from piglets of less than 1 weekof age (3). These results suggest that in the pig intestinecertain 08:KX105 strains may produce a surface antigen thatlacks genetic homology with the F6 structural subunit butthat is antigenically related to it. On the other hand, it ispossible that our F6 (987P) antiserum contained antibodiesdirected against surface components other than the F6(987P) structural subunit (6, 32). Such antibodies would reactwith the antigenically related antigens that are expressed onthe surface of 08:KX105 strains when they are grown in thepig intestine. These hypotheses are presently under investi-gation (Broes et al., in press).We have thus demonstrated that (i) there is a strong

correlation between serogroup 08:KX105 and pathogenicityfor piglets, (ii) the 08:KX105 strain population is structurallyrelatively heterogeneous, and (iii) 08:KX105 strains do notpossess the genes encoding the F6 (987P) fimbrial coloniza-tion factor. The mechanisms by which 08:KX105 strainscolonize the small intestine during infection and the nature of

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2408 BROES ET AL.

the F6 reaction demonstrated by these strains in the intes-tines of infected piglets remain to be elucidated.

ACKNOWLEDGMENTS

This study was supported by grant MMV-85-C-1150 from theConseil des Recherches et Services Agricoles du Québec.We thank Clarisse Desautels, Céline Forget, Etienne Jacquemin,

Richard Bourassa, Linda Leblanc, and Pierre Demers for technicalassistance. We are grateful to Ida and Frits Orskov (InternationalEscherichia and Klebsiella Centre, Copenhagen, Denmark) forserotyping the strains, Pierre Pohl and Paul Lintermans (InstitutNational de Recherches Vétérinaires, Brussels, Belgium), HarleyW. Moon (National Animal Disease Center, Ames, Iowa), and FritsK. De Graaf (Vrije Universiteit, Amsterdam, The Netherlands) forproviding strains, antiserum, or both.

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