JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1125-11310095-1137/90/061125-07$02.00/0Copyright © 1990, American Society for Microbiology

Comparison of Restriction Endonuclease Analysis andPhenotypic Typing Methods for Differentiation

of Yersinia enterocolitica IsolatesGEORG KAPPERUD,1,2* TRULS NESBAKKEN,2 STOJANKA ALEKSIC,3

AND HENRI H. MOLLARETDepartment of Bacteriology, National Institute of Public Health, 0462 Oslo 4,1 and Department ofFood Hygiene, The Norwegian College of Veterinary Medicine, POB 8146 Dep, 0033 Oslo 1,2

Norway; Hygienisches Institut, 2000 Hamburg, Federal Republic of Germany3; andUnité d'Ecologie Bactérienne, Institut Pasteur, 75724 Paris Cedex 15, France4

Received 6 September 1989/Accepted 28 February 1990

Restriction endonuclease analysis of chromosomal DNA (REAC) was used to study polymorphism inrestriction fragment patterns among Yersinia enterocolitica isolates belonging to serogroups 03, 05,27, 08,09, 013, and 021. Using the enzyme HaeIII and electrophoresis on thin (0.75-mm) vertical 5% polyacrylamidegels, we were able to distinguish at least 22 DNA fragment patterns among the 72 strains examined. The methodshowed the greatest discriminatory power with regard to serogroup 08, within which as many as 10 differentDNA fragment patterns were detected among the 16 strains examined. Compared with 08, serogroups 03 and09 were relatively homogeneous with regard to REAC patterns. The discriminatory power of the method wascompared with H-antigen typing, biotyping, phage typing, antimicrobial susceptibility typing, and restrictionenzyme analysis of the virulence plasmid (REAP), by means of Simpson's index of diversity. The results showedthat REAC and REAP constitute an effective supplement or alternative to conventional phenotypic methods fortracing epidemiologically related isolates of Y. enterocolitica. Our finding that human and porcine isolatesexhibited the same REAC, REAP, and H-antigen patterns provides additional support for the hypothesis thatpigs play an important role in the epidemiology of human Y. enterocolitica infection.

Various laboratory methods have been employed in at-tempts to differentiate Yersinia enterocolitica isolates, withthe objective of developing epidemiological tools. The mostwidely used methods have relied upon phenotypic charac-teristics such as biochemical properties (biotyping) (29), 0and H antigens (serotyping) (2, 27), antibiotic susceptibility(antibiogram typing) (3, 8), and bacteriophage lysis patterns(phage typing) (3, 17, 20). The recent advances in genetechnology have provided several new typing methods thatare based on characterization of the genotype. These meth-ods include plasmid profile analysis, analysis of restrictionendonuclease cleavage patterns, and the use of DNA orRNA probes (5, 15, 24, 26). Restriction enzyme analysis ofchromosomal DNA (REAC) (DNA fingerprinting) has beenused successfully in epidemiological studies of several bac-terial infections (5, 6, 11, 13, 25). In a previous investigation,we employed restriction enzyme analysis to investigate thestructural variability of the 40- to 50-megadalton virulenceplasmid of Y. enterocolitica (19). The DNA fragment profileswere found to vary, not only between serogroups but alsoamong plasmids from strains with the same serogroup affil-iation. The results indicated that structural variability in theplasmid concerned may be used to differentiate Y. enteroco-litica isolates that would otherwise be indistinguishable.

In this investigation, we used an improved REAC methodwhich enabled simple and reproducible differentiation of Y.enterocolitica isolates. The method was compared with fourtraditional phenotypic typing systems and with restrictionanalysis of the virulence plasmid, by using a numerical indexto monitor relative discriminatory power.Although there is strong indirect evidence that pigs are an

* Corresponding author.

important reservoir for human infection with Y. enteroco-litica (10, 23), we are not aware of a single reported case inwhich pigs or pork products were directly incriminated asthe source of infection. In our previous investigation, wefound that porcine and human isolates harbored virulenceplasmids with identical restriction patterns (19). The presentinvestigation offered an opportunity to compare human andporcine isolates by using REAC.

MATERIALS AND METHODSBacterial isolates. A total of 72 Yersinia isolates belonging

to serogroups 03, 09, 08, 05,27, 013, and 021 wereselected for this study (Table 1). The isolates were obtainedfrom human patients, animals, food, and environmentalsources in 14 countries in Europe, North America, and Asia.Of the 72 isolates studied, 68 belonged to Y. enterocolitica,while 4 belonged to other Yersinia species.

Selection of plasmid-bearing and plasmid-cured variants.All strains were examined for the presence of the 40- to50-megadalton virulence plasmid, as described previously(19). The plasmid was cured by repeated cultivation onmagnesium oxalate agar (21) as detailed elsewhere (19). Allstrains were checked for plasmid DNA content before fur-ther analysis. Phenotypic characterization of the strains andrestriction enzyme analysis of plasmid DNA (REAP) werecarried out with plasmid-bearing variants. REAC was basedon isogenic plasmid-cured derivatives. Restriction enzymeanalyses were conducted at the National Institute of PublicHealth, Oslo, Norway. Phenotypic characterization wasperformed at the Hygiene Institute, Hamburg, Federal Re-public of Germany, except for phage typing, which wascarried out at the Pasteur Institute, Paris, France.REAC. Plasmid-cured bacteria were grown overnight in 5

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TABLE 1. Phenotypic and genotypic characterization of 72 Yersinia strains

Strain Source Country DonorL H antigen Biovarb Phagevar REAP REAC Antibiogramc

Serogroup 08 (n = 16)8081A2635NY81-68NY81-71WA182417001821NY81-85NY81-87YE10.121YE665YE653FRI-YE1FRI-YE3FRI-YE10

Serogroup 021 (n = 1):YE737

Human USAHuman USAHuman USAHuman USAHuman USAHuman USAHuman USAHuman USAHuman USAHuman USAHuman CanadaHuman CanadaHuman CanadaPig USAPig USAPig USA

Human Canada

B befiQ befiN befiN befiQ befiv0 befi0 befi0 befivN befivN befiR befivD befiD befivE befivE befivE befiv

1B Xz A 1 (1), (6), (8), (15), (16)1B Xz B 2 (6)1B Xz C 3 (10), 151B Xz C 4 (6)1B Xz D 5 (6)1B Xz F 6 (6),151B Xz F 6 (6),151B Xz F 6 (10), 151B Xz F 6 (6), (15), (16)1B Xz F 7 (1), (6)1B Xz B 8 (6),151B Xz C 9 (6), (16)1B Xz E 10 (1), (6)1B Xz B 2 (6),151B Xz B 21B Xz B 2 (6), (15)

D befi 1B Xz G il (1), (4), (6)

Serogroup 013a, 13b (n = 1): Primate CanadaYE886

D abi 1B Xo H 12 (1), (4), (6)

Serogroup 03 (n = 33)29C-4629C-43a29C-33201/861389/861084/86Y2aY89Y310Y763Y772Y764Y325P77421603a2713-TKS4147366882658258a8246MCH697MCH700YE11.131YE11.137YE751YE859PA11472SW13123M254PA11400SW13711M388

Serogroup 09 (n = 11)3315a352078948125YE0997OULU

Human NorwayHuman NorwayHuman NorwayHuman NorwayHuman NorwayHuman NorwayPig NorwayPig NorwayPig NorwayPig NorwayPig NorwayPig NorwayPork NorwayPig SwedenPig DenmarkHuman FinlandHuman FinlandHuman FinlandHuman FranceHuman SpainHuman YugoslaviaHuman CanadaHuman CanadaHuman CanadaHuman CanadaHuman CanadaPork CanadaHuman JapanPig JapanPork JapanHuman JapanPig JapanPork Japan

Human NetherlandsHuman NetherlandsHuman FranceHuman FranceHuman CanadaHuman Finland

H abcH abcH abcH abcH d

H cL acL acL abcvL _dL cL abcL abcG abcC abcP abcM abcM abcJ abcJ abcJ abcI abcI abcR bcR abcD abcD abcF abcF acF acF _dF d

F d

K abvK abJ abJ abD abvP abv

4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 VIII4 IXb4 IXb4 IXb4 IXb4 IXb4 IXb4 VIII4 VIII4 VIII3 Il3 Il3 Il

I 13 1,4,6I 13 1,4,6I 13 1, (2), 4, 6, (7), 15I 13 1, 4, 6, (16)I 13 1, 4J 13 1, (2), 4, 6, 15, (16)I 13 1, 4, 6I 13 1, 4, 6, 15I 13 1, 4, 6I 13 1, 4, 6I 13 1, 4, 6, (12)I 1De 1, 4, 6, 15, (16)I 13 1, 4, 6, 15I 13 1, 4, 6, 15I 1Ye 1, 4, 6, 15I 1Ye 1, 4, 6K 13e (1), 4K 13e 1, 4, 6, (14), 15I 13 1, 4, 6, 15I 1e 1, 4, 6I 1e 1, 4, 6I 1e 1, 4, 6, (7), 15I 1e 1, 4, 5, 6, (7), 15, (16)I 13Y 1, 4, 6, (7), 15I 1e 1, 4, 6, 15I 14 (6), (16)I 1e 1, 4, (6), 15I 1e 1, 4, 6I 1e (1), 6I 1e (1), 4, (6)I 15 1, 3, 4, (5), 6I 15 1, 3, 4, (5), 6, 15I 15 1, 3, 4, 5, 6

2 X3 L 16 1, 4, 6, (7), 152 X3 L 16 1, 4, (6)2 X3 L 16e 1, 2, 6, 13, 152 X3 L 16 1, 4, 62 X3 L 16e (1), (2), 3, 4, 6, 152 X3 L 16 (1), (4), (6)

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TABLE 1-Continued

Strain Source Country Donor' H antigen Biovarb Phagevar REAP REAC Antibiogramc

7877 Human Belgium J ab 2 X3 L 16e 1, 3, 4, 6, 15PA177 Human Japan F abc 2 X3 L 16e 1, 4, 6, 15W827 Pig Belgium S ab 2 X3 L 16e 1, (4), 6, (13), 15W828 Pig Belgium S ab 2 X3 L 16e 1, 4, (6), (13), 15W829 Pig Belgium S ab 2 X3 L 16e 1, 2, 4, (6), (13)

Serogroup 05,27 (n = 5)SW14391 Pig Japan F abc 2 Xz M 17 3D113 Dog Japan F abc 2 Xz M 17 (1), 3, 4, (5), (6), 7, 9,

11, 13, 15PA9436 Human Japan F abc 2 Xz N 17 (1), 3, 4, (5)YE771 Human Canada D bcv 2 Xz N 17 3, 5, 6YE873 Pork Canada D abc 2 Xz N 17 (1), 4, (5), (6), 7, 15,

16

Serogroup 03, atypicalstrains (n = 5)

87-36/82 Human Germany A u YMf XI -g 18 1, 3, 4, 5, (6)8018 Rodent Norway L abcd 1A xI 19 (1), (6)176-36/80 Sewage Germany A p2 YFh XI -g 20 (1), (2), 4, 6357-36/85 Water Germany A q Y' x I 21 (1), (6)Y332 Pork Norway L __d YKi Xo -g 22 1, 4, 6, (7)a The strains (except our own isolates [L; 12, 18]) were received from the following scientists: S. Aleksic (A), I. Bolin (B), S. G. Christensen (C), J. Devenish

(D), M. P. Doyle (E), H. Fukushima (F), B. Hurvell (G), J. Lassen (H), C-J. Lian (I), H. H. Mollaret (J), J. Oosterom (K), L. Pulkkinen (M), T. J. Quan (N),M. Shayegani (0), C. Sundqvist (P), B. Swaminathan (Q), S. Toma (R), and G. Wauters (S).

b According to the revised scheme of Wauters et al. (29).c Numbers indicate resistance against various antibiotics. Numbers in parentheses indicate intermediate resistance. 1, Ampicillin; 2, apalcillin; 3, cefaclor; 4,

cefazolin; 5, cefoxitin; 6, cefsulodin; 7, ceftazidime; 8, gentamicin; 9, kanamycin; 10, sisomicin; 11, streptomycin; 12, chloramphenicol; 13, phosphomycin; 14,polymyxin; 15, sulfonamide; 16, tetracycline.

d Nonmotile.e Strains showing minor deviations from a particular REAC pattern (see Fig. 2 and 3).f YM, Yersinia mollaretii (28).g Lacks the virulence plasmid.h YF, Yersinia frederiksenii.YK, Yersinia kristensenii.

i YI, Yersinia intermedia.

ml of Trypticase soy broth (BBL Microbiology Systems,Cockeysville, Md.) containing 0.6% yeast extract. The bac-terial density was then adjusted spectrophotometrically to anoptical density of 0.95 at 660 nm (Hitachi 101 spectropho-tometer). Chromosomal DNA was extracted by using amodification of the rapid small-scale technique for isolationof plasmid DNA proposed by Maniatis et al. (14). Samples(1.0 ml) of bacterial suspension were centrifuged, and thebacteria were lysed with lysozyme (14). After 5 min at roomtemperature, 200 ,tl of a solution containing 1% sodiumdodecyl sulfate, 0.1 ml NaCI, and 0.1 M Tris base (pH 8.5)were added. After 5 min on ice, 1.0 ,ul ofproteinase K (1.5%)was added and the lysates were incubated at 37°C for 1 h.The lysates were then washed two times with phenol-chloroform, and DNA was precipitated with ethanol (14).The resulting DNA preparations were digested with restric-tion enzymes under conditions recommended by the supplier(Toyobo Co. Ltd., Osaka, Japan). Results obtained by usingthe enzymes RsrII, SmaI, EcoRI, BamHI, RsaI, HaeIII, andTaqI were compared in a pilot study. Electrophoresis wasperformed at various voltages and durations in agarose gel(0.5 to 3.0%) and in polyacrylamide gel (3.2 to 6.0%). Thebest resolution was obtained with HaeIII when the digestswere subjected to electrophoresis for 19 h at 70 V on avertical 5% polyacrylamide gel (0.75 mm thick), in Tris-borate-EDTA buffer, by using a Mini-Protean Il slab gel cell(Bio-Rad Laboratories, Richmond, Calif.). Consequently, allisolates were examined by this method.Another pilot study demonstrated that HaeIII digests of

total DNA from plasmid-bearing and plasmid-cured isogenic

FIG. 1. Comparison of restriction endonuclease (HaeIII) cleav-age patterns of DNA from plasmid-bearing and plasmid-curedisogenic derivatives of two Y. enterocolitica strains. Lanes: Athrough C, strain 29C-43; D through F, strain 8081. The followingcleavage patterns are presented: plasmid DNA (lanes A and D), totalDNA from plasmid-bearing variants (lanes B and E), and chromo-somal DNA from isogenic plasmid-cured derivatives (lanes C andF). The size marker at the left is a 1-kilobase DNA ladder. Thefollowing fragments are indicated by arrows: 5.1, 3.1, 2.0, 1.6, and1.0 kilobases.

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derivatives resulted in significantly different DNA fragmentpatterns (Fig. 1). Ail analyses were consequently carried outwith plasmid-cured variants. A 1-kilobase DNA ladder (Be-thesda Research Laboratories, Inc., Gaithersburg, Md.) wasincluded as a size marker on each gel.REAP. REAP was performed by using the enzymes EcoRI

and BamHI as described previously (19). The results havebeen presented elsewhere (19). The present investigationincluded two strains from Finland (4147 and 3668) showing aREAP pattern which differed from those described in ourprevious work (22).

Phenotypic characterization. All strains were serotypedby slide agglutination by using specific antisera preparedagainst 60 0 antigens and 38 H antigens, as describedelsewhere (1, 2). Biotyping was performed according to therevised biotyping scheme proposed by Wauters et al. (29).Phage typing was carried out by using the scheme publishedby Nicolle (20). Ail strains were tested for susceptibilityto 29 different antimicrobial agents on Iso-Sensitest agar(Oxoid Ltd., Basingstoke, Hampshire, England) by thestandardized agar diffusion method (4). Commercial antibi-otic disks were purchased from Oxoid Ltd. Plates wereincubated at 28°C for 48 h, and zones of complete inhibitionwere measured to the nearest millimeter. The isolates wereclassified as resistant, sensitive, or intermediate, accordingto standard criteria (Methoden zur Empfindlichkeitsprùfungvon bakteriellen Krankheitserregern [ausser Mykobakte-rien] gegen Chemotherapeutica, DIN 58940, Teil 1-6, Beuth-Verlag, Berlin 1979-1987). The following antibiotics weretested: penicillins-ampicillin, apalcillin, azlocillin, me-zlocillin, and piperacillin; cephalosporins-cefaclor, cefazo-lin, cefmenoxime, cefoperazone, cefoxitin, cefsulodin,ceftazidime, ceftriaxone, and moxalactam; aminoglyco-sides-gentamicin, kanamycin, neomycin, sisomicin, andstreptomycin; quinolones-enoxacin and ofloxacin; andother antibiotics-chloramphenicol, deblastone, nitrofuran-toin, phosphomycin, polymyxin, sulfamethoxazole-trimeth-oprim, sulfonamide, and tetracycline.

Discrimination index. The discriminatory power of eachtyping method or combination of methods was compared bySimpson's index of diversity, as suggested by Hunter andGaston (9). This index estimates the probability that twounrelated strains sampled at random from the test populationwill be placed into different typing groups.

RESULTS

REAC. REAC using the enzyme HaeIII allowed 22 dis-tinct restriction patterns to be distinguished among the 72Yersinia strains examined (Table 1; Fig. 2). Each REACpattern was serogroup specific. The DNA profiles varied,not only between serogroups but also between isolatesbelonging to the same serogroup (Table 1). REAC proved tohave the greatest discriminatory power within serogroup 08,in which as many as 10 distinct profiles were differentiated inthe 16 strains investigated. Compared with serogroup 08,serogroups 03 and 09 were relatively homogeneous withregard to REAC patterns.With regard to two of the REAC profiles (profiles 13 and

16), a not inconsiderable variability was demonstrated in thata number of strains showed slight yet distinct deviationsfrom the main pattern (Fig. 3). This variability could not beexplained by the presence of plasmids other than the viru-lence plasmid. However, strains with such slight variationswere difficult to group in relation to each other. In an attemptto resolve this problem, chromosomal DNA from all strains

I3 4 8 9 l 1 il 12

S 19l :2 21 22

FIG. 2. Restriction endonuclease (HaeIII) cleavage patterns ofchromosomal DNA from Yersinia strains with different serogroupaffiliations. The numbers 1 to 22 represent the 22 different REACpatterns detected (Table 1). Panels A and B show serogroups 08(lanes 1 through 10), 021 (lane 11), and 013 (lane 12). The isolatesare represented as follows. Lanes: 1, 8081; 2, A2635; 3, NY81-68; 4,NY81-71; 5, WA; 6, 1824; 7, NY81-87; 8, YE10.121; 9, YE665; 10,YE653; 11, YE737; and 12, YE886. Panel C shows serogroups 03(lanes 13 through 15), 09 (lane 16), and 05,27 (lane 17). The isolatesare represented as follows. Lanes: 13, 29C-46; 14, YE751; 15,SW13711; 16, 70ULU; and 17, YE771. Panel D shows atypical 03isolates (lanes 18 through 22). The isolates are represented asfollows. Lanes: 18, 87-36/82; 19, 8018; 20, 176-36/80; 21, 357-36/85;and 22, Y332. Unlabeled lanes are size markers (1-kilobase DNAladder; Fig. 1).

concerned was analyzed by using RsaI and RsaI-HaeIII(double digested). Again, a slight yet reproducible variabilitywas observed (Fig. 3), but the strains were still difficult togroup.

A B C D E

FIG. 3. Restriction endonuclease (HaeIII) cleavage patterns ofchromosomal DNA from five Y. enterocolitica serogroup 03 iso-lates which exhibit slight yet reproducible deviations from REACpattern 13 (Table 1). The isolates are represented as follows. Lanes:A, 21603a; B, 4147; C, 8265; D, 8258a; and E, 8246. Size markers areat right (Fig. 1).

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TABLE 2. Comparison of discriminatory power of Y. enterocolitica typing methods

Serogroup 08 (n = 16) Serogroup 03 (n = 33)a Serogroup 09 (n = 11) Total (n = 72)Typing method__ ___

No. of typesb DIC No. of types DI No. of types DI No. of types DI

Antibiogram 9 0.908 19 0.902 il 1.000 43 0.965REAC 10 0.900 4 0.623 2 0.509 22 0.903H-antigen typing 2 0.533 6 0.608 3 0.564 16 0.840REAP 6 0.808 3 0.174 1 0.000 15 0.793Phage typing 1 0.000 3 0.443 1 0.000 7 0.770Biotypingd 1 0.000 2 0.170 1 0.000 9 0.722

a Only the pathogenic variants (biovars 4 and 3) were included.b Nontypable isolates were considered as a separate type. REAC patterns showing minor deviations from a particular REAC pattern (13' and 16e, Table 1) were

considered as distinct types.C DI, Simpson's index of diversity (9).d Y. enterocolitica was biotyped by the method of Wauters et al. (29). Other Yersinia species were considered as separate biotypes.

The REAC profile of each isolate included in this studywas evaluated at least twice and on different gels. All 72isolates showed identical patterns on initial and repeatanalyses except YE886, which was incompletely digested onseveral occasions. Thus, the reproducibility of the REACpatterns was 98.6%. To further investigate the reproducibil-ity, seven subcultures of the same strain (PA11400) were setup on different days. Each subculture was then processedseparately by the method described above and subjected toelectrophoresis in the same gel. No significant differencesbetween the seven REAC patterns could be detected.Comparison of genotypic and phenotypic typing methods.

The discriminatory power of a typing method is its ability todistinguish between unrelated strains. It is determined by thenumber of types defined by the method in question and therelative frequency of these types (9). Simpson's index ofdiversity, which presents these two facets of discriminationas a single numerical value, was used to compare REACwith the other methods employed (Table 2). Although theisolates examined were not a random selection or repre-sentative of the wide diversity encountered within Y. entero-colitica, this index allows objective assessment of discrimi-natory power within the population of test isolates whichincluded the most important pathogenic serogroups. Sinceserogroup affiliation was used as the basis for preselection ofthe isolates, 0-antigen typing could not be compared withthe other methods. Of the six methods compared, antimicro-bial susceptibility typing proved to have the greatest overalldiscriminatory power, followed by REAC, H-antigen typing,REAP, phage typing, and biotyping (in decreasing order ofdiscrimination) (Table 2).Comparison of human and porcine isolates. Part of the

study involved 23 isolates from pigs and pork, all belongingto serogroups associated with disease in humans. No differ-ences in REAC patterns between porcine isolates and humanclinical isolates belonging to serogroups 03, 08, or 09 couldbe demonstrated (Table 1). In order to investigate thephenotypic and genotypic diversity among strains from thesame country, 13 isolates of serogroup 03, biovar 4, phage-var VIII from human patients and pigs in Norway wereincluded. Although these strains could not be distinguishedfrom each other by REAC, considerable variations in H-antigen pattern and antibiotic resistance were found, and onestrain also exhibited a deviating REAP profile (Table 1).

DISCUSSION

Restriction fragment patterns of chromosomal DNA ortotal DNA preparations are difficult to compare because ofthe large number of bands produced. We achieved good

separation of the heaviest fragments by using the four-basecutter HaeIII and electrophoresis on thin polyacrylamidegels. Results were easy to interpret and reproducible. Com-parison of the discriminatory power of six phenotypic andgenotypic typing methods revealed that REAC ranked sec-ond, above H-antigen typing but below antimicrobial sus-ceptibility testing. However, these results are valid only forthe selection of isolates included in this study and are notnecessarily representative of the wide diversity of strainsfound within the whole species.An advantage of typing methods based on the investiga-

tion of genotype is that the same set of reagents andequipment can be used (only small modifications are re-quired) for many different bacteria (15, 26). In contrast,phage typing requires a battery of phages and indicatorstrains and is standardized for only a few species of bacteria.Serotyping likewise demands a large number of antiserawhich are difficult and expensive to prepare and standardize.Another advantage of genotypic methods is that they avoidproblems associated with gene expression in vitro (15, 26).Many phenotypic characteristics vary depending on certainenvironmental or culture-related conditions, a circumstancewhich gives rise to diverging results between different labo-ratories.Of the two genotypic methods employed, REAP was the

easiest to perform. Moreover, the results obtained by thismethod were easier to interpret than the complicated DNAfragment patterns obtained by REAC. However, in thisstudy, a considerably better discrimination between isolateswas achieved with REAC than with REAP, although thelatter method involved the use of two restriction enzymeswhile REAC was based on the use of only one. Anotheradvantage of REAC is that the method can be applied evenif the bacterium to be studied has accidentally lost thevirulence plasmid, while REAP, obviously, can only beemployed if the plasmid is present. However, since presenceof the virulence plasmid has a significant effect on the REACpatterns obtained (Fig. 1), one should ensure that all strainsbeing compared are either plasmid cured or plasmid bearing.REAC and REAP both permitted differentiation of isolates

which were indistinguishable by the phenotypic methodsemployed. On the other hand, it was in many cases possibleto achieve a finer degree of differentiation of isolates with thesame REAC or REAP patterns by using one or more of thephenotypic methods (Table 1). As regards Y. enterocolitica,geographic origin, ecologic characteristics, and pathogenicsignificance are closely correlated with certain combinationsof serogroup, biovar, and phagevar (16). These classicalphenotypic parameters thus provide valuable information on

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the strains, even though they should be combined with othermethods such as REAC, REAP, or H-antigen typing ifoptimal differentiation is to be achieved.The discriminatory power of the genotypic methods was

greatest within serogroup 08 (Table 2). Since this serogroupis homogeneous with regard to biovar and phagevar, therehas long been a need for methods which permit a finer degreeof differentiation in epidemiological investigations (3). A newbacteriophage typing system which allows greater differen-tiation of 08 strains has been described (3). The results ofour investigation show that restriction enzyme analysispresents itself as an effective alternative or supplement totraditional methods, including phage typing, for the analysisof serogroup 08.Most isolates of Y. enterocolitica possess H antigens

consisting of several subfactors. In a previous study, 117serovars within Y. enterocolitica were defined on the basis ofH-antigen patterns. The H antigens are monophasic andremain stable after repeated subcultivation and after storageas stab cultures (2). The great variability in H antigensdemonstrated in the present and previous studies alloweddifferentiation of strains which could not be distinguished byany of the other typing methods employed. Even though thepreparation and standardization of specific antisera are bothtime-consuming and costly, H-antigen typing thus neverthe-less is a valuable supplement to 0-antigen typing and bio-chemical characterization of Y. enterocolitica in epidemio-logical investigations.

Antimicrobial susceptibility testing is a highly standard-ized method employed in a large number of laboratories.However, susceptibility patterns may be influenced by localantibiotic usage, which may make differentiation of outbreakstrains from epidemiologically unrelated strains difficult. Ofthe six typing methods which were compared in this study,antimicrobial susceptibility testing showed the greatest vari-ability in all the serogroups investigated (Tables 1 and 2).However, on the basis of the investigation of strains fromthree outbreaks, Baker and Farmer (3) concluded that eventhough antimicrobial susceptibility testing contributed to thedifferentiation of the isolates, results were less reliable thanthose obtained by serotyping and phage typing. The differ-ences in antibiograms reported in the present study shouldbe interpreted with caution. Although the definition of resis-tance versus susceptibility is based on significant differencesin zone sizes, a number of isolates showed intermediatevalues, indicating that the antibiograms are not necessarilycompletely reproducible.Three strains of serogroup 08 included in this study were

originally isolated in connection with an outbreak of food-borne yersiniosis in a summer camp in Sullivan County,New York, in 1981 (strains 1700, 1821, and 1824) (M.Shayegani, personal communication). It is interesting thatwhile all strains had the same REAC and REAP profiles, onestrain exhibited a deviating H-antigen pattern and a slightlydifferent antibiogram, compared with the other two (Table1).

In a parallel study, we employed multilocus enzymeelectrophoresis to investigate allelic variations in the chro-mosomal genomes of the same strains included in this study(7). Results revealed only slight variability between strainsof the same serogroup. The method would have given anoverall discrimination index of 0.752, indicating it to be lesssuitable for tracing epidemiologically related Y. enteroco-litica strains.From an epidemiological point of view, it is interesting

that isolates from pigs showed the same REAC and REAP

patterns as human isolates, regardless of the serogroupaffiliation. The same H antigens, biovars, phagevars, andantibiograms were also represented among isolates fromboth humans and pigs. Even though the relative frequency ofdifferent variants in pigs and humans was not systematicallyinvestigated, these observations nevertheless reinforce oursupposition that pigs constitute an important source ofhuman infection.

ACKNOWLEDGMENTS

The antibiotic susceptibility testing was kindly performed by A.Katz. We thank Bill Davies and Henning S0rum for helpful discus-sions. The technical assistance of Traute Vardund, Kari Dommars-nes, and Berit Gamnes is gratefully acknowledged.

This work was supported by grants from the Norwegian Councilfor Agricultural Research.

LITERATURE CITED1. Aleksic, S., and J. Bockemuhl. 1987. Diagnostic importance of

H-antigens in Yersinia enterocolitica and other Yersinia species.Contrib. Microbiol. Immunol. 9:279-284.

2. Aleksic, S., J. Bockemuhl, and F. Lange. 1986. Studies on theserology of flagellar antigens of Yersinia enterocolitica andrelated Yersinia species. Zentralbl. Bakteriol. Mikrobiol. Hyg.Ser. A 261:299-310.

3. Baker, P. M., and J. J. Farmer III. 1982. New bacteriophagetyping system for Yersinia enterocolitica, Yersinia kristensenii,Yersiniafrederiksenii, and Yersinia intermedia: correlation withserotyping, biotyping, and antibiotic susceptibility. J. Clin.Microbiol. 15:491-502.

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