Vol. 28, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1125-1131
0095-1137/90/061125-07$02.00/0 Copyright © 1990, American Society for Microbiology
Comparison of Restriction Endonuclease Analysis and Phenotypic Typing Methods for Differentiation of Yersinia enterocolitica Isolates GEORG KAPPERUD,1,2* TRULS NESBAKKEN,2 STOJANKA ALEKSIC,3 AND HENRI H. MOLLARET Department of Bacteriology, National Institute of Public Health, 0462 Oslo 4,1 and Department of Food Hygiene, The Norwegian College of Veterinary Medicine, POB 8146 Dep, 0033 Oslo 1,2 Norway; Hygienisches Institut, 2000 Hamburg, Federal Republic of Germany3; and Unité 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 in restriction 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% polyacrylamide gels, we were able to distinguish at least 22 DNA fragment patterns among the 72 strains examined. The method showed the greatest discriminatory power with regard to serogroup 08, within which as many as 10 different DNA fragment patterns were detected among the 16 strains examined. Compared with 08, serogroups 03 and 09 were relatively homogeneous with regard to REAC patterns. The discriminatory power of the method was compared with H-antigen typing, biotyping, phage typing, antimicrobial susceptibility typing, and restriction enzyme analysis of the virulence plasmid (REAP), by means of Simpson's index of diversity. The results showed that REAC and REAP constitute an effective supplement or alternative to conventional phenotypic methods for tracing epidemiologically related isolates of Y. enterocolitica. Our finding that human and porcine isolates exhibited the same REAC, REAP, and H-antigen patterns provides additional support for the hypothesis that pigs play an important role in the epidemiology of human Y. enterocolitica infection. Various laboratory methods have been employed in attempts to differentiate Yersinia enterocolitica isolates, with the objective of developing epidemiological tools. The most
important reservoir for human infection with Y. enterocolitica (10, 23), we are not aware of a single reported case in which pigs or pork products were directly incriminated as the source of infection. In our previous investigation, we found that porcine and human isolates harbored virulence plasmids with identical restriction patterns (19). The present investigation offered an opportunity to compare human and porcine isolates by using REAC.
widely used methods have relied upon phenotypic characteristics such as biochemical properties (biotyping) (29), 0 and H antigens (serotyping) (2, 27), antibiotic susceptibility (antibiogram typing) (3, 8), and bacteriophage lysis patterns (phage typing) (3, 17, 20). The recent advances in gene technology have provided several new typing methods that are based on characterization of the genotype. These methods include plasmid profile analysis, analysis of restriction endonuclease cleavage patterns, and the use of DNA or RNA probes (5, 15, 24, 26). Restriction enzyme analysis of chromosomal DNA (REAC) (DNA fingerprinting) has been used successfully in epidemiological studies of several bacterial infections (5, 6, 11, 13, 25). In a previous investigation, we employed restriction enzyme analysis to investigate the structural variability of the 40- to 50-megadalton virulence plasmid of Y. enterocolitica (19). The DNA fragment profiles were found to vary, not only between serogroups but also among plasmids from strains with the same serogroup affiliation. The results indicated that structural variability in the plasmid concerned may be used to differentiate Y. enterocolitica isolates that would otherwise be indistinguishable. In this investigation, we used an improved REAC method which enabled simple and reproducible differentiation of Y. enterocolitica isolates. The method was compared with four traditional phenotypic typing systems and with restriction analysis of the virulence plasmid, by using a numerical index to monitor relative discriminatory power. Although there is strong indirect evidence that pigs are an *
MATERIALS AND METHODS Bacterial isolates. A total of 72 Yersinia isolates belonging to serogroups 03, 09, 08, 05,27, 013, and 021 were selected for this study (Table 1). The isolates were obtained from human patients, animals, food, and environmental sources 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- to 50-megadalton virulence plasmid, as described previously (19). The plasmid was cured by repeated cultivation on magnesium oxalate agar (21) as detailed elsewhere (19). All strains were checked for plasmid DNA content before further analysis. Phenotypic characterization of the strains and restriction enzyme analysis of plasmid DNA (REAP) were carried out with plasmid-bearing variants. REAC was based on isogenic plasmid-cured derivatives. Restriction enzyme analyses were conducted at the National Institute of Public Health, Oslo, Norway. Phenotypic characterization was performed at the Hygiene Institute, Hamburg, Federal Republic of Germany, except for phage typing, which was carried out at the Pasteur Institute, Paris, France. REAC. Plasmid-cured bacteria were grown overnight in 5
Corresponding author. 1125
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TABLE 1. Phenotypic and genotypic characterization of 72 Yersinia strains DonorL H antigen Biovarb Phagevar REAP REAC Country Source
Strain
Serogroup 08 (n = 16) 8081
Antibiogramc
N Q 0 0 0 N N R D D E E E
befi befi befi befi befiv befi befi befiv befiv befi befiv befi befiv befiv befiv befiv
1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B
Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz
A B C C D F F F F F B C E B B B
1 2 3 4 5 6 6 6 6 7 8 9 10 2 2 2
(1), (6), (8), (15), (16) (6) (10), 15 (6) (6) (6),15 (6),15 (10), 15 (6), (15), (16) (1), (6) (6),15 (6), (16) (1), (6) (6),15
Canada
D
befi
1B
Xz
G
il
(1), (4), (6)
Primate
Canada
D
abi
1B
Xo
H
12
(1), (4), (6)
Human Human Human Human Human Human
Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Sweden Denmark Finland Finland Finland France
H H H H
abc abc abc abc
VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII
13 13 13 13 13 13 13 13 13 13 13
VIII VIII VIII Il Il Il
I I I I I J I I I I I I I I I I K K I I I I I I I I I I I I I I I
1,4,6 1,4,6 1, (2), 4, 6, (7), 15 1, 4, 6, (16) 1, 4 1, (2), 4, 6, 15, (16) 1, 4, 6 1, 4, 6, 15 1, 4, 6 1, 4, 6 1, 4, 6, (12) 1, 4, 6, 15, (16) 1, 4, 6, 15 1, 4, 6, 15 1, 4, 6, 15 1, 4, 6 (1), 4 1, 4, 6, (14), 15 1, 4, 6, 15 1, 4, 6 1, 4, 6 1, 4, 6, (7), 15 1, 4, 5, 6, (7), 15, (16) 1, 4, 6, (7), 15 1, 4, 6, 15 (6), (16) 1, 4, (6), 15 1, 4, 6 (1), 6 (1), 4, (6) 1, 3, 4, (5), 6 1, 3, 4, (5), 6, 15 1, 3, 4, 5, 6
X3 X3 X3 X3 X3 X3
L L L L L L
16 16 16e 16 16e 16
A2635 NY81-68 NY81-71 WA 1824 1700 1821 NY81-85 NY81-87 YE10.121 YE665 YE653 FRI-YE1 FRI-YE3 FRI-YE10
Human Human Human Human Human Human Human Human Human Human Human Human Human Pig Pig Pig
USA USA USA USA USA USA USA USA USA USA Canada Canada Canada USA USA USA
Serogroup 021 (n = 1): YE737
Human
Serogroup 013a, 13b (n = 1):
B
Q N
(6), (15)
YE886
Serogroup 03 (n = 33) 29C-46 29C-43a 29C-33 201/86 1389/86 1084/86 Y2a Y89 Y310 Y763 Y772 Y764 Y325 P774 21603a 2713-TKS 4147 3668 8265 8258a 8246 MCH697 MCH700 YE11.131 YE11.137 YE751 YE859 PA11472 SW13123 M254 PA11400 SW13711 M388
Serogroup 09 (n 3315a 3520 7894 8125 YE099
7OULU
=
Pig Pig Pig Pig Pig Pig Pork Pig Pig Human Human Human Human Human Human Human Human Human Human Human Pork Human
Pig Pork
Spain Yugoslavia Canada Canada Canada Canada Canada Canada Japan
Pig Pork
Japan Japan Japan Japan Japan
Human Human Human Human Human Human
Netherlands Netherlands France France Canada Finland
Human
H
d
H L L L L L L L G C P M M J J J I I R R D D F F F F
c ac ac abcv
F
d
F
d
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3
K K
abv ab ab ab abv abv
2 2 2 2 2 2
_d
c
abc abc abc abc abc abc abc abc abc abc abc abc bc abc abc abc abc ac ac _d
IXb IXb
IXb IXb IXb
IXb
1De 13 13
1Ye 1Ye 13e 13e 13 1e 1e 1e 1e
13Y 1e 14 1e 1e 1e 1e 15 15 15
11) J J D P
1, 4, 6, (7), 15 1, 4, (6) 1, 2, 6, 13, 15 1, 4, 6 (1), (2), 3, 4, 6, 15 (1), (4), (6) Continued on following page
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TABLE 1-Continued Strain
7877 PA177 W827 W828 W829 Serogroup 05,27 (n = 5) SW14391 D113 PA9436 YE771 YE873
Source
Country
Donor'
H antigen
Biovarb
Phagevar
REAP
REAC
Antibiogramc
Human Human Pig Pig Pig
Belgium Japan Belgium Belgium Belgium
J F S S S
ab abc ab ab ab
2 2 2 2 2
X3 X3 X3 X3 X3
L L L L L
16e 16e 16e 16e 16e
1, 1, 1, 1, 1,
Pig Dog
Japan Japan
F F
abc abc
2 2
Xz Xz
M M
17 17
3
Human Human Pork
Japan Canada Canada
F D D
abc bcv abc
2 2 2
Xz Xz Xz
N N N
17 17 17
3, 4, 6, 15 4, 6, 15 (4), 6, (13), 15 4, (6), (13), 15 2, 4, (6), (13)
(1), 3, 4, (5), (6), 7, 9, 11, 13, 15 (1), 3, 4, (5) 3, 5, 6 (1), 4, (5), (6), 7, 15, 16
Serogroup 03, atypical strains (n = 5) 87-36/82 8018 176-36/80 357-36/85 Y332
Human Rodent Sewage Water Pork
Germany Norway Germany Germany Norway
A L A A L
u abcd p2 q __d
YMf
XI xI XI xI Xo
1A YFh Y'
-g -g
18 19 20 21 22
1, 3, 4, 5, (6) (1), (6) (1), (2), 4, 6 (1), (6) 1, 4, 6, (7)
YKi -g 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 bacterial density was then adjusted spectrophotometrically to an optical density of 0.95 at 660 nm (Hitachi 101 spectrophotometer). Chromosomal DNA was extracted by using a modification of the rapid small-scale technique for isolation of plasmid DNA proposed by Maniatis et al. (14). Samples (1.0 ml) of bacterial suspension were centrifuged, and the bacteria were lysed with lysozyme (14). After 5 min at room temperature, 200 ,tl of a solution containing 1% sodium dodecyl sulfate, 0.1 ml NaCI, and 0.1 M Tris base (pH 8.5) were added. After 5 min on ice, 1.0 ,ul of proteinase K (1.5%) was added and the lysates were incubated at 37°C for 1 h. The lysates were then washed two times with phenolchloroform, and DNA was precipitated with ethanol (14). The resulting DNA preparations were digested with restriction enzymes under conditions recommended by the supplier (Toyobo Co. Ltd., Osaka, Japan). Results obtained by using the enzymes RsrII, SmaI, EcoRI, BamHI, RsaI, HaeIII, and
TaqI were compared in a pilot study. Electrophoresis was performed at various voltages and durations in agarose gel (0.5 to 3.0%) and in polyacrylamide gel (3.2 to 6.0%). The best resolution was obtained with HaeIII when the digests were subjected to electrophoresis for 19 h at 70 V on a vertical 5% polyacrylamide gel (0.75 mm thick), in Trisborate-EDTA buffer, by using a Mini-Protean Il slab gel cell (Bio-Rad Laboratories, Richmond, Calif.). Consequently, all isolates were examined by this method. Another pilot study demonstrated that HaeIII digests of total DNA from plasmid-bearing and plasmid-cured isogenic
ib ib
FIG. 1. Comparison of restriction endonuclease (HaeIII) cleavage patterns of DNA from plasmid-bearing and plasmid-cured isogenic derivatives of two Y. enterocolitica strains. Lanes: A through C, strain 29C-43; D through F, strain 8081. The following cleavage patterns are presented: plasmid DNA (lanes A and D), total DNA from plasmid-bearing variants (lanes B and E), and chromosomal DNA from isogenic plasmid-cured derivatives (lanes C and F). The size marker at the left is a 1-kilobase DNA ladder. The following fragments are indicated by arrows: 5.1, 3.1, 2.0, 1.6, and 1.0 kilobases.
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derivatives resulted in significantly different DNA fragment patterns (Fig. 1). Ail analyses were consequently carried out with plasmid-cured variants. A 1-kilobase DNA ladder (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was included as a size marker on each gel. REAP. REAP was performed by using the enzymes EcoRI and BamHI as described previously (19). The results have been presented elsewhere (19). The present investigation included two strains from Finland (4147 and 3668) showing a REAP pattern which differed from those described in our previous work (22). Phenotypic characterization. All strains were serotyped by slide agglutination by using specific antisera prepared against 60 0 antigens and 38 H antigens, as described elsewhere (1, 2). Biotyping was performed according to the revised biotyping scheme proposed by Wauters et al. (29). Phage typing was carried out by using the scheme published by Nicolle (20). Ail strains were tested for susceptibility to 29 different antimicrobial agents on Iso-Sensitest agar (Oxoid Ltd., Basingstoke, Hampshire, England) by the standardized agar diffusion method (4). Commercial antibiotic disks were purchased from Oxoid Ltd. Plates were incubated at 28°C for 48 h, and zones of complete inhibition were measured to the nearest millimeter. The isolates were classified as resistant, sensitive, or intermediate, according to standard criteria (Methoden zur Empfindlichkeitsprùfung von bakteriellen Krankheitserregern [ausser Mykobakterien] gegen Chemotherapeutica, DIN 58940, Teil 1-6, BeuthVerlag, Berlin 1979-1987). The following antibiotics were tested: penicillins-ampicillin, apalcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins-cefaclor, cefazolin, cefmenoxime, cefoperazone, cefoxitin, cefsulodin, ceftazidime, ceftriaxone, and moxalactam; aminoglycosides-gentamicin, kanamycin, neomycin, sisomicin, and streptomycin; quinolones-enoxacin and ofloxacin; and other antibiotics-chloramphenicol, deblastone, nitrofurantoin, phosphomycin, polymyxin, sulfamethoxazole-trimethoprim, sulfonamide, and tetracycline. Discrimination index. The discriminatory power of each typing method or combination of methods was compared by Simpson's index of diversity, as suggested by Hunter and Gaston (9). This index estimates the probability that two unrelated strains sampled at random from the test population will be placed into different typing groups. RESULTS REAC. REAC using the enzyme HaeIII allowed 22 distinct restriction patterns to be distinguished among the 72 Yersinia strains examined (Table 1; Fig. 2). Each REAC pattern was serogroup specific. The DNA profiles varied, not only between serogroups but also between isolates belonging to the same serogroup (Table 1). REAC proved to have the greatest discriminatory power within serogroup 08, in which as many as 10 distinct profiles were differentiated in the 16 strains investigated. Compared with serogroup 08, serogroups 03 and 09 were relatively homogeneous with regard to REAC patterns. With regard to two of the REAC profiles (profiles 13 and 16), a not inconsiderable variability was demonstrated in that a number of strains showed slight yet distinct deviations from the main pattern (Fig. 3). This variability could not be explained by the presence of plasmids other than the virulence plasmid. However, strains with such slight variations were difficult to group in relation to each other. In an attempt to resolve this problem, chromosomal DNA from all strains
I3
8 9 l
4
S 19 l
1 il 12
:2 21 2 2
FIG. 2. Restriction endonuclease (HaeIII) cleavage patterns of chromosomal DNA from Yersinia strains with different serogroup affiliations. The numbers 1 to 22 represent the 22 different REAC patterns detected (Table 1). Panels A and B show serogroups 08 (lanes 1 through 10), 021 (lane 11), and 013 (lane 12). The isolates are 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 isolates are represented as follows. Lanes: 13, 29C-46; 14, YE751; 15, SW13711; 16, 70ULU; and 17, YE771. Panel D shows atypical 03 isolates (lanes 18 through 22). The isolates are represented as follows. 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 DNA ladder; Fig. 1).
concerned was analyzed by using RsaI and RsaI-HaeIII (double digested). Again, a slight yet reproducible variability was observed (Fig. 3), but the strains were still difficult to group.
A
B C
D
E
FIG. 3. Restriction endonuclease (HaeIII) cleavage patterns of chromosomal DNA from five Y. enterocolitica serogroup 03 isolates which exhibit slight yet reproducible deviations from REAC pattern 13 (Table 1). The isolates are represented as follows. Lanes: A, 21603a; B, 4147; C, 8265; D, 8258a; and E, 8246. Size markers are at
right (Fig. 1).
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TABLE 2. Comparison of discriminatory power of Y. enterocolitica typing methods Serogroup 03 (n = 33)a
Serogroup 08 (n = 16) Typing method__
Antibiogram REAC H-antigen typing REAP Phage typing Biotypingd
Total (n = 72)
Serogroup 09 (n = 11)
___
No. of typesb
DIC
No. of types
DI
No. of types
DI
No. of types
DI
9 10 2 6 1 1
0.908 0.900 0.533 0.808 0.000 0.000
19 4 6 3 3 2
0.902 0.623 0.608 0.174 0.443 0.170
il 2 3 1 1 1
1.000 0.509 0.564 0.000 0.000 0.000
43 22 16 15 7 9
0.965 0.903 0.840 0.793 0.770 0.722
a Only the pathogenic variants (biovars 4 and 3) were included. 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. b
The REAC profile of each isolate included in this study was evaluated at least twice and on different gels. All 72 isolates showed identical patterns on initial and repeat analyses except YE886, which was incompletely digested on several occasions. Thus, the reproducibility of the REAC patterns was 98.6%. To further investigate the reproducibility, seven subcultures of the same strain (PA11400) were set up on different days. Each subculture was then processed separately by the method described above and subjected to electrophoresis in the same gel. No significant differences between the seven REAC patterns could be detected. Comparison of genotypic and phenotypic typing methods. The discriminatory power of a typing method is its ability to distinguish between unrelated strains. It is determined by the number of types defined by the method in question and the relative frequency of these types (9). Simpson's index of diversity, which presents these two facets of discrimination as a single numerical value, was used to compare REAC with the other methods employed (Table 2). Although the isolates examined were not a random selection or representative of the wide diversity encountered within Y. enterocolitica, this index allows objective assessment of discriminatory power within the population of test isolates which included the most important pathogenic serogroups. Since serogroup affiliation was used as the basis for preselection of the isolates, 0-antigen typing could not be compared with the other methods. Of the six methods compared, antimicrobial susceptibility typing proved to have the greatest overall discriminatory power, followed by REAC, H-antigen typing, REAP, phage typing, and biotyping (in decreasing order of discrimination) (Table 2). Comparison of human and porcine isolates. Part of the study involved 23 isolates from pigs and pork, all belonging to serogroups associated with disease in humans. No differences in REAC patterns between porcine isolates and human clinical isolates belonging to serogroups 03, 08, or 09 could be demonstrated (Table 1). In order to investigate the phenotypic and genotypic diversity among strains from the same country, 13 isolates of serogroup 03, biovar 4, phagevar VIII from human patients and pigs in Norway were included. Although these strains could not be distinguished from each other by REAC, considerable variations in Hantigen pattern and antibiotic resistance were found, and one strain also exhibited a deviating REAP profile (Table 1). DISCUSSION Restriction fragment patterns of chromosomal DNA or total DNA preparations are difficult to compare because of the large number of bands produced. We achieved good
separation of the heaviest fragments by using the four-base cutter HaeIII and electrophoresis on thin polyacrylamide gels. Results were easy to interpret and reproducible. Comparison of the discriminatory power of six phenotypic and genotypic typing methods revealed that REAC ranked second, above H-antigen typing but below antimicrobial susceptibility testing. However, these results are valid only for the selection of isolates included in this study and are not necessarily representative of the wide diversity of strains found within the whole species. An advantage of typing methods based on the investigation of genotype is that the same set of reagents and equipment can be used (only small modifications are required) for many different bacteria (15, 26). In contrast, phage typing requires a battery of phages and indicator strains and is standardized for only a few species of bacteria. Serotyping likewise demands a large number of antisera which are difficult and expensive to prepare and standardize. Another advantage of genotypic methods is that they avoid problems associated with gene expression in vitro (15, 26). Many phenotypic characteristics vary depending on certain environmental or culture-related conditions, a circumstance which gives rise to diverging results between different laboratories. Of the two genotypic methods employed, REAP was the easiest to perform. Moreover, the results obtained by this method were easier to interpret than the complicated DNA fragment patterns obtained by REAC. However, in this study, a considerably better discrimination between isolates was achieved with REAC than with REAP, although the latter method involved the use of two restriction enzymes while REAC was based on the use of only one. Another advantage of REAC is that the method can be applied even if the bacterium to be studied has accidentally lost the virulence plasmid, while REAP, obviously, can only be employed if the plasmid is present. However, since presence of the virulence plasmid has a significant effect on the REAC patterns obtained (Fig. 1), one should ensure that all strains being compared are either plasmid cured or plasmid bearing. REAC and REAP both permitted differentiation of isolates which were indistinguishable by the phenotypic methods employed. On the other hand, it was in many cases possible to achieve a finer degree of differentiation of isolates with the same REAC or REAP patterns by using one or more of the phenotypic methods (Table 1). As regards Y. enterocolitica, geographic origin, ecologic characteristics, and pathogenic significance are closely correlated with certain combinations of serogroup, biovar, and phagevar (16). These classical phenotypic parameters thus provide valuable information on
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the strains, even though they should be combined with other methods such as REAC, REAP, or H-antigen typing if optimal differentiation is to be achieved. The discriminatory power of the genotypic methods was greatest within serogroup 08 (Table 2). Since this serogroup is homogeneous with regard to biovar and phagevar, there has long been a need for methods which permit a finer degree of differentiation in epidemiological investigations (3). A new bacteriophage typing system which allows greater differentiation of 08 strains has been described (3). The results of our investigation show that restriction enzyme analysis presents itself as an effective alternative or supplement to traditional methods, including phage typing, for the analysis of serogroup 08. Most isolates of Y. enterocolitica possess H antigens consisting of several subfactors. In a previous study, 117 serovars within Y. enterocolitica were defined on the basis of H-antigen patterns. The H antigens are monophasic and remain stable after repeated subcultivation and after storage as stab cultures (2). The great variability in H antigens demonstrated in the present and previous studies allowed differentiation of strains which could not be distinguished by any of the other typing methods employed. Even though the preparation and standardization of specific antisera are both time-consuming and costly, H-antigen typing thus nevertheless is a valuable supplement to 0-antigen typing and biochemical characterization of Y. enterocolitica in epidemiological investigations. Antimicrobial susceptibility testing is a highly standardized method employed in a large number of laboratories. However, susceptibility patterns may be influenced by local antibiotic usage, which may make differentiation of outbreak strains from epidemiologically unrelated strains difficult. Of the six typing methods which were compared in this study, antimicrobial susceptibility testing showed the greatest variability in all the serogroups investigated (Tables 1 and 2). However, on the basis of the investigation of strains from three outbreaks, Baker and Farmer (3) concluded that even though antimicrobial susceptibility testing contributed to the differentiation of the isolates, results were less reliable than those obtained by serotyping and phage typing. The differences in antibiograms reported in the present study should be interpreted with caution. Although the definition of resistance versus susceptibility is based on significant differences in zone sizes, a number of isolates showed intermediate values, indicating that the antibiograms are not necessarily completely reproducible. Three strains of serogroup 08 included in this study were originally isolated in connection with an outbreak of foodborne yersiniosis in a summer camp in Sullivan County, New York, in 1981 (strains 1700, 1821, and 1824) (M. Shayegani, personal communication). It is interesting that while all strains had the same REAC and REAP profiles, one strain exhibited a deviating H-antigen pattern and a slightly different antibiogram, compared with the other two (Table 1).
In a parallel study, we employed multilocus enzyme electrophoresis to investigate allelic variations in the chromosomal genomes of the same strains included in this study (7). Results revealed only slight variability between strains of the same serogroup. The method would have given an overall discrimination index of 0.752, indicating it to be less suitable for tracing epidemiologically related Y. enterocolitica strains. From an epidemiological point of view, it is interesting that isolates from pigs showed the same REAC and REAP
J. CLIN. MICROBIOL.
patterns as human isolates, regardless of the serogroup affiliation. The same H antigens, biovars, phagevars, and antibiograms were also represented among isolates from both humans and pigs. Even though the relative frequency of different variants in pigs and humans was not systematically investigated, these observations nevertheless reinforce our supposition that pigs constitute an important source of human infection. ACKNOWLEDGMENTS The antibiotic susceptibility testing was kindly performed by A. Katz. We thank Bill Davies and Henning S0rum for helpful discussions. The technical assistance of Traute Vardund, Kari Dommarsnes, and Berit Gamnes is gratefully acknowledged. This work was supported by grants from the Norwegian Council for Agricultural Research. LITERATURE CITED 1. 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 the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species. Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 261:299-310. 3. Baker, P. M., and J. J. Farmer III. 1982. New bacteriophage typing system for Yersinia enterocolitica, Yersinia kristensenii, Yersiniafrederiksenii, and Yersinia intermedia: correlation with serotyping, biotyping, and antibiotic susceptibility. J. Clin. Microbiol. 15:491-502. 4. Balows, A. 1976. Performance standards for antimicrobial disc susceptibility tests as used in clinical laboratories, p. 138-155. In A. Balows (ed.), Current techniques for antibiotic susceptibility testing. Charles C Thomas, Publisher, Springfield, III. 5. Bjorvatn, B., and B.-E. Kristiansen. 1985. Molecular epidemiology of bacterial infections. Clin. Lab. Med. 5:437-445. 6. Bradbury, W. C., A. D. Pearson, M. A. Marko, R. V. Congi, and J. L. Penner. 1984. Investigation of a Campylobacter jejuni outbreak by serotyping and chromosomal restriction endonuclease analysis. J. Clin. Microbiol. 19:342-346. 7. Caugant, D. A., S. Aleksic, H. H. Mollaret, R. K. Selander, and G. Kapperud. 1989. Clonal diversity and relationships among strains of Yersinia enterocolitica. J. Clin. Microbiol. 27:26782683. 8. Cornelis, G. 1981. Antibiotic resistance in Yersinia enterocolitica, p. 55-71. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 9. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466. 10. Hurvell, B. 1981. Zoonotic Yersinia enterocolitica infection: host range, clinical manifestations, and transmission between animals and man, p. 145-160. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 11. Kaper, J. B., H. B. Bradford, N. C. Roberts, and S. Falkow. 1982. Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast. J. Clin. Microbiol. 16:129-134. 12. Kapperud, G. 1981. Survey on the reservoirs of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in Scandinavia. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 89:29-35. 13. Kristiansen, B.-E., B. S0rensen, B. Bjorvatn, E. S. Falk, E. Fosse, K. Bryn, L. O. Fr0holm, P. Gaustad, and K. B0vre. 1986. An outbreak of group B meningococcal disease: tracing the causative strain of Neisseria meningitidis by DNA fingerprinting. J. Clin. Microbiol. 23:764-767. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, p. 368-369. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Mayer, L. W. 1988. Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance. Clin. Microbiol. Rev. 1:228-243.
VOL. 28, 1990 16. Mollaret, H. H., H. Bercovier, and J. A. Alonso. 1979. Summary of the data received at the WHO Reference Center for Yersinia enterocolitica. Contrib. Microbiol. Immunol. 5:174-184. 17. Moliaret, H. H., and P. Nicolle. 1965. Sur la fréquence de la lysogénie dans l'espèce nouvelle Yersinia enterocolitica. C.R. Acad. Sci. 260:1027-1029. 18. Nesbakken, T., B. Gondrosen, and G. Kapperud. 1985. Investigation of Yersinia enterocolitica, Yersinia enterocolitica-like bacteria, and thermotolerant campylobacters in Norwegian pork products. Int. J. Food Microbiol. 1:311-320. 19. Nesbakken, T., G. Kapperud, H. S0rum, and K. Dommarsnes. 1987. Structural variability of 40-50 Mdal virulence plasmids from Yersinia enterocolitica. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 95:167-173. 20. Nicoile, P. 1973. Yersinia enterocolitica, p. 377-387. In H. Rische (ed.), Lysotypie und andere spezielle epidemiologische Laboratoriums-methoden. VEB Gustav Fisher Verlag, Jena, German Democratic Republic. 21. Perry, R. D., and R. R. Brubaker. 1983. Vwa+ phenotype of Yersinia enterocolitica. Infect. Immun. 40:166-171. 22. Pulkkinen, L., I. Granberg, K. Granfors, and A. Toivanen. 1986. Restriction map of virulence plasmid in Yersinia enterocolitica 0:3. Plasmid 16:225-227.
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23. Tauxe, R. V., J. Vandepitte, G. Wauters, S. M. Martin, V. Goossens, P. De Mol, R. Van Noyen, and G. Thiers. 1987. Yersinia enterocolitica infections and pork: the missing link. Lancet i:1129-1132. 24. Tenover, F. C. 1988. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbiol. Rev. 1:82-101. 25. Tompkins, L. S., N. J. Troup, T. Woods, W. Bibb, and R. M. McKinney. 1987. Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis. J. Clin. Microbiol. 25:1875-1880. 26. Wachsmuth, K. 1985. Genotypic approaches to the diagnosis of bacterial infections: plasmid analyses and gene probes. Infect. Control 6:100-109. 27. Wauters, G. 1981. Antigens of Yersinia enterocolitica, p. 41-53. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 28. Wauters, G., M. Janssens, A. G. Steigerwalt, and D. J. Brenner. 1988. Yersinia mollaretii sp. nov. and Yersinia bercovieri sp. nov., formerly called Yersinia enterocolitica biogroups 3A and 3B. Int. J. Syst. Bacteriol. 38:424-429. 29. Wauters, G., K. Kandolo, and M. Janssens. 1987. Revised biogrouping scheme of Yersinia enterocolitica. Contrib. Microbiol. Immunol. 9:14-21.
JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1125-1131
0095-1137/90/061125-07$02.00/0 Copyright © 1990, American Society for Microbiology
Comparison of Restriction Endonuclease Analysis and Phenotypic Typing Methods for Differentiation of Yersinia enterocolitica Isolates GEORG KAPPERUD,1,2* TRULS NESBAKKEN,2 STOJANKA ALEKSIC,3 AND HENRI H. MOLLARET Department of Bacteriology, National Institute of Public Health, 0462 Oslo 4,1 and Department of Food Hygiene, The Norwegian College of Veterinary Medicine, POB 8146 Dep, 0033 Oslo 1,2 Norway; Hygienisches Institut, 2000 Hamburg, Federal Republic of Germany3; and Unité 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 in restriction 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% polyacrylamide gels, we were able to distinguish at least 22 DNA fragment patterns among the 72 strains examined. The method showed the greatest discriminatory power with regard to serogroup 08, within which as many as 10 different DNA fragment patterns were detected among the 16 strains examined. Compared with 08, serogroups 03 and 09 were relatively homogeneous with regard to REAC patterns. The discriminatory power of the method was compared with H-antigen typing, biotyping, phage typing, antimicrobial susceptibility typing, and restriction enzyme analysis of the virulence plasmid (REAP), by means of Simpson's index of diversity. The results showed that REAC and REAP constitute an effective supplement or alternative to conventional phenotypic methods for tracing epidemiologically related isolates of Y. enterocolitica. Our finding that human and porcine isolates exhibited the same REAC, REAP, and H-antigen patterns provides additional support for the hypothesis that pigs play an important role in the epidemiology of human Y. enterocolitica infection. Various laboratory methods have been employed in attempts to differentiate Yersinia enterocolitica isolates, with the objective of developing epidemiological tools. The most
important reservoir for human infection with Y. enterocolitica (10, 23), we are not aware of a single reported case in which pigs or pork products were directly incriminated as the source of infection. In our previous investigation, we found that porcine and human isolates harbored virulence plasmids with identical restriction patterns (19). The present investigation offered an opportunity to compare human and porcine isolates by using REAC.
widely used methods have relied upon phenotypic characteristics such as biochemical properties (biotyping) (29), 0 and H antigens (serotyping) (2, 27), antibiotic susceptibility (antibiogram typing) (3, 8), and bacteriophage lysis patterns (phage typing) (3, 17, 20). The recent advances in gene technology have provided several new typing methods that are based on characterization of the genotype. These methods include plasmid profile analysis, analysis of restriction endonuclease cleavage patterns, and the use of DNA or RNA probes (5, 15, 24, 26). Restriction enzyme analysis of chromosomal DNA (REAC) (DNA fingerprinting) has been used successfully in epidemiological studies of several bacterial infections (5, 6, 11, 13, 25). In a previous investigation, we employed restriction enzyme analysis to investigate the structural variability of the 40- to 50-megadalton virulence plasmid of Y. enterocolitica (19). The DNA fragment profiles were found to vary, not only between serogroups but also among plasmids from strains with the same serogroup affiliation. The results indicated that structural variability in the plasmid concerned may be used to differentiate Y. enterocolitica isolates that would otherwise be indistinguishable. In this investigation, we used an improved REAC method which enabled simple and reproducible differentiation of Y. enterocolitica isolates. The method was compared with four traditional phenotypic typing systems and with restriction analysis of the virulence plasmid, by using a numerical index to monitor relative discriminatory power. Although there is strong indirect evidence that pigs are an *
MATERIALS AND METHODS Bacterial isolates. A total of 72 Yersinia isolates belonging to serogroups 03, 09, 08, 05,27, 013, and 021 were selected for this study (Table 1). The isolates were obtained from human patients, animals, food, and environmental sources 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- to 50-megadalton virulence plasmid, as described previously (19). The plasmid was cured by repeated cultivation on magnesium oxalate agar (21) as detailed elsewhere (19). All strains were checked for plasmid DNA content before further analysis. Phenotypic characterization of the strains and restriction enzyme analysis of plasmid DNA (REAP) were carried out with plasmid-bearing variants. REAC was based on isogenic plasmid-cured derivatives. Restriction enzyme analyses were conducted at the National Institute of Public Health, Oslo, Norway. Phenotypic characterization was performed at the Hygiene Institute, Hamburg, Federal Republic of Germany, except for phage typing, which was carried out at the Pasteur Institute, Paris, France. REAC. Plasmid-cured bacteria were grown overnight in 5
Corresponding author. 1125
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TABLE 1. Phenotypic and genotypic characterization of 72 Yersinia strains DonorL H antigen Biovarb Phagevar REAP REAC Country Source
Strain
Serogroup 08 (n = 16) 8081
Antibiogramc
N Q 0 0 0 N N R D D E E E
befi befi befi befi befiv befi befi befiv befiv befi befiv befi befiv befiv befiv befiv
1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B
Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz Xz
A B C C D F F F F F B C E B B B
1 2 3 4 5 6 6 6 6 7 8 9 10 2 2 2
(1), (6), (8), (15), (16) (6) (10), 15 (6) (6) (6),15 (6),15 (10), 15 (6), (15), (16) (1), (6) (6),15 (6), (16) (1), (6) (6),15
Canada
D
befi
1B
Xz
G
il
(1), (4), (6)
Primate
Canada
D
abi
1B
Xo
H
12
(1), (4), (6)
Human Human Human Human Human Human
Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Norway Sweden Denmark Finland Finland Finland France
H H H H
abc abc abc abc
VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII
13 13 13 13 13 13 13 13 13 13 13
VIII VIII VIII Il Il Il
I I I I I J I I I I I I I I I I K K I I I I I I I I I I I I I I I
1,4,6 1,4,6 1, (2), 4, 6, (7), 15 1, 4, 6, (16) 1, 4 1, (2), 4, 6, 15, (16) 1, 4, 6 1, 4, 6, 15 1, 4, 6 1, 4, 6 1, 4, 6, (12) 1, 4, 6, 15, (16) 1, 4, 6, 15 1, 4, 6, 15 1, 4, 6, 15 1, 4, 6 (1), 4 1, 4, 6, (14), 15 1, 4, 6, 15 1, 4, 6 1, 4, 6 1, 4, 6, (7), 15 1, 4, 5, 6, (7), 15, (16) 1, 4, 6, (7), 15 1, 4, 6, 15 (6), (16) 1, 4, (6), 15 1, 4, 6 (1), 6 (1), 4, (6) 1, 3, 4, (5), 6 1, 3, 4, (5), 6, 15 1, 3, 4, 5, 6
X3 X3 X3 X3 X3 X3
L L L L L L
16 16 16e 16 16e 16
A2635 NY81-68 NY81-71 WA 1824 1700 1821 NY81-85 NY81-87 YE10.121 YE665 YE653 FRI-YE1 FRI-YE3 FRI-YE10
Human Human Human Human Human Human Human Human Human Human Human Human Human Pig Pig Pig
USA USA USA USA USA USA USA USA USA USA Canada Canada Canada USA USA USA
Serogroup 021 (n = 1): YE737
Human
Serogroup 013a, 13b (n = 1):
B
Q N
(6), (15)
YE886
Serogroup 03 (n = 33) 29C-46 29C-43a 29C-33 201/86 1389/86 1084/86 Y2a Y89 Y310 Y763 Y772 Y764 Y325 P774 21603a 2713-TKS 4147 3668 8265 8258a 8246 MCH697 MCH700 YE11.131 YE11.137 YE751 YE859 PA11472 SW13123 M254 PA11400 SW13711 M388
Serogroup 09 (n 3315a 3520 7894 8125 YE099
7OULU
=
Pig Pig Pig Pig Pig Pig Pork Pig Pig Human Human Human Human Human Human Human Human Human Human Human Pork Human
Pig Pork
Spain Yugoslavia Canada Canada Canada Canada Canada Canada Japan
Pig Pork
Japan Japan Japan Japan Japan
Human Human Human Human Human Human
Netherlands Netherlands France France Canada Finland
Human
H
d
H L L L L L L L G C P M M J J J I I R R D D F F F F
c ac ac abcv
F
d
F
d
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3
K K
abv ab ab ab abv abv
2 2 2 2 2 2
_d
c
abc abc abc abc abc abc abc abc abc abc abc abc bc abc abc abc abc ac ac _d
IXb IXb
IXb IXb IXb
IXb
1De 13 13
1Ye 1Ye 13e 13e 13 1e 1e 1e 1e
13Y 1e 14 1e 1e 1e 1e 15 15 15
11) J J D P
1, 4, 6, (7), 15 1, 4, (6) 1, 2, 6, 13, 15 1, 4, 6 (1), (2), 3, 4, 6, 15 (1), (4), (6) Continued on following page
REAC AND REAP OF YERSINIA SPP.
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1127
TABLE 1-Continued Strain
7877 PA177 W827 W828 W829 Serogroup 05,27 (n = 5) SW14391 D113 PA9436 YE771 YE873
Source
Country
Donor'
H antigen
Biovarb
Phagevar
REAP
REAC
Antibiogramc
Human Human Pig Pig Pig
Belgium Japan Belgium Belgium Belgium
J F S S S
ab abc ab ab ab
2 2 2 2 2
X3 X3 X3 X3 X3
L L L L L
16e 16e 16e 16e 16e
1, 1, 1, 1, 1,
Pig Dog
Japan Japan
F F
abc abc
2 2
Xz Xz
M M
17 17
3
Human Human Pork
Japan Canada Canada
F D D
abc bcv abc
2 2 2
Xz Xz Xz
N N N
17 17 17
3, 4, 6, 15 4, 6, 15 (4), 6, (13), 15 4, (6), (13), 15 2, 4, (6), (13)
(1), 3, 4, (5), (6), 7, 9, 11, 13, 15 (1), 3, 4, (5) 3, 5, 6 (1), 4, (5), (6), 7, 15, 16
Serogroup 03, atypical strains (n = 5) 87-36/82 8018 176-36/80 357-36/85 Y332
Human Rodent Sewage Water Pork
Germany Norway Germany Germany Norway
A L A A L
u abcd p2 q __d
YMf
XI xI XI xI Xo
1A YFh Y'
-g -g
18 19 20 21 22
1, 3, 4, 5, (6) (1), (6) (1), (2), 4, 6 (1), (6) 1, 4, 6, (7)
YKi -g 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 bacterial density was then adjusted spectrophotometrically to an optical density of 0.95 at 660 nm (Hitachi 101 spectrophotometer). Chromosomal DNA was extracted by using a modification of the rapid small-scale technique for isolation of plasmid DNA proposed by Maniatis et al. (14). Samples (1.0 ml) of bacterial suspension were centrifuged, and the bacteria were lysed with lysozyme (14). After 5 min at room temperature, 200 ,tl of a solution containing 1% sodium dodecyl sulfate, 0.1 ml NaCI, and 0.1 M Tris base (pH 8.5) were added. After 5 min on ice, 1.0 ,ul of proteinase K (1.5%) was added and the lysates were incubated at 37°C for 1 h. The lysates were then washed two times with phenolchloroform, and DNA was precipitated with ethanol (14). The resulting DNA preparations were digested with restriction enzymes under conditions recommended by the supplier (Toyobo Co. Ltd., Osaka, Japan). Results obtained by using the enzymes RsrII, SmaI, EcoRI, BamHI, RsaI, HaeIII, and
TaqI were compared in a pilot study. Electrophoresis was performed at various voltages and durations in agarose gel (0.5 to 3.0%) and in polyacrylamide gel (3.2 to 6.0%). The best resolution was obtained with HaeIII when the digests were subjected to electrophoresis for 19 h at 70 V on a vertical 5% polyacrylamide gel (0.75 mm thick), in Trisborate-EDTA buffer, by using a Mini-Protean Il slab gel cell (Bio-Rad Laboratories, Richmond, Calif.). Consequently, all isolates were examined by this method. Another pilot study demonstrated that HaeIII digests of total DNA from plasmid-bearing and plasmid-cured isogenic
ib ib
FIG. 1. Comparison of restriction endonuclease (HaeIII) cleavage patterns of DNA from plasmid-bearing and plasmid-cured isogenic derivatives of two Y. enterocolitica strains. Lanes: A through C, strain 29C-43; D through F, strain 8081. The following cleavage patterns are presented: plasmid DNA (lanes A and D), total DNA from plasmid-bearing variants (lanes B and E), and chromosomal DNA from isogenic plasmid-cured derivatives (lanes C and F). The size marker at the left is a 1-kilobase DNA ladder. The following fragments are indicated by arrows: 5.1, 3.1, 2.0, 1.6, and 1.0 kilobases.
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derivatives resulted in significantly different DNA fragment patterns (Fig. 1). Ail analyses were consequently carried out with plasmid-cured variants. A 1-kilobase DNA ladder (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was included as a size marker on each gel. REAP. REAP was performed by using the enzymes EcoRI and BamHI as described previously (19). The results have been presented elsewhere (19). The present investigation included two strains from Finland (4147 and 3668) showing a REAP pattern which differed from those described in our previous work (22). Phenotypic characterization. All strains were serotyped by slide agglutination by using specific antisera prepared against 60 0 antigens and 38 H antigens, as described elsewhere (1, 2). Biotyping was performed according to the revised biotyping scheme proposed by Wauters et al. (29). Phage typing was carried out by using the scheme published by Nicolle (20). Ail strains were tested for susceptibility to 29 different antimicrobial agents on Iso-Sensitest agar (Oxoid Ltd., Basingstoke, Hampshire, England) by the standardized agar diffusion method (4). Commercial antibiotic disks were purchased from Oxoid Ltd. Plates were incubated at 28°C for 48 h, and zones of complete inhibition were measured to the nearest millimeter. The isolates were classified as resistant, sensitive, or intermediate, according to standard criteria (Methoden zur Empfindlichkeitsprùfung von bakteriellen Krankheitserregern [ausser Mykobakterien] gegen Chemotherapeutica, DIN 58940, Teil 1-6, BeuthVerlag, Berlin 1979-1987). The following antibiotics were tested: penicillins-ampicillin, apalcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins-cefaclor, cefazolin, cefmenoxime, cefoperazone, cefoxitin, cefsulodin, ceftazidime, ceftriaxone, and moxalactam; aminoglycosides-gentamicin, kanamycin, neomycin, sisomicin, and streptomycin; quinolones-enoxacin and ofloxacin; and other antibiotics-chloramphenicol, deblastone, nitrofurantoin, phosphomycin, polymyxin, sulfamethoxazole-trimethoprim, sulfonamide, and tetracycline. Discrimination index. The discriminatory power of each typing method or combination of methods was compared by Simpson's index of diversity, as suggested by Hunter and Gaston (9). This index estimates the probability that two unrelated strains sampled at random from the test population will be placed into different typing groups. RESULTS REAC. REAC using the enzyme HaeIII allowed 22 distinct restriction patterns to be distinguished among the 72 Yersinia strains examined (Table 1; Fig. 2). Each REAC pattern was serogroup specific. The DNA profiles varied, not only between serogroups but also between isolates belonging to the same serogroup (Table 1). REAC proved to have the greatest discriminatory power within serogroup 08, in which as many as 10 distinct profiles were differentiated in the 16 strains investigated. Compared with serogroup 08, serogroups 03 and 09 were relatively homogeneous with regard to REAC patterns. With regard to two of the REAC profiles (profiles 13 and 16), a not inconsiderable variability was demonstrated in that a number of strains showed slight yet distinct deviations from the main pattern (Fig. 3). This variability could not be explained by the presence of plasmids other than the virulence plasmid. However, strains with such slight variations were difficult to group in relation to each other. In an attempt to resolve this problem, chromosomal DNA from all strains
I3
8 9 l
4
S 19 l
1 il 12
:2 21 2 2
FIG. 2. Restriction endonuclease (HaeIII) cleavage patterns of chromosomal DNA from Yersinia strains with different serogroup affiliations. The numbers 1 to 22 represent the 22 different REAC patterns detected (Table 1). Panels A and B show serogroups 08 (lanes 1 through 10), 021 (lane 11), and 013 (lane 12). The isolates are 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 isolates are represented as follows. Lanes: 13, 29C-46; 14, YE751; 15, SW13711; 16, 70ULU; and 17, YE771. Panel D shows atypical 03 isolates (lanes 18 through 22). The isolates are represented as follows. 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 DNA ladder; Fig. 1).
concerned was analyzed by using RsaI and RsaI-HaeIII (double digested). Again, a slight yet reproducible variability was observed (Fig. 3), but the strains were still difficult to group.
A
B C
D
E
FIG. 3. Restriction endonuclease (HaeIII) cleavage patterns of chromosomal DNA from five Y. enterocolitica serogroup 03 isolates which exhibit slight yet reproducible deviations from REAC pattern 13 (Table 1). The isolates are represented as follows. Lanes: A, 21603a; B, 4147; C, 8265; D, 8258a; and E, 8246. Size markers are at
right (Fig. 1).
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TABLE 2. Comparison of discriminatory power of Y. enterocolitica typing methods Serogroup 03 (n = 33)a
Serogroup 08 (n = 16) Typing method__
Antibiogram REAC H-antigen typing REAP Phage typing Biotypingd
Total (n = 72)
Serogroup 09 (n = 11)
___
No. of typesb
DIC
No. of types
DI
No. of types
DI
No. of types
DI
9 10 2 6 1 1
0.908 0.900 0.533 0.808 0.000 0.000
19 4 6 3 3 2
0.902 0.623 0.608 0.174 0.443 0.170
il 2 3 1 1 1
1.000 0.509 0.564 0.000 0.000 0.000
43 22 16 15 7 9
0.965 0.903 0.840 0.793 0.770 0.722
a Only the pathogenic variants (biovars 4 and 3) were included. 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. b
The REAC profile of each isolate included in this study was evaluated at least twice and on different gels. All 72 isolates showed identical patterns on initial and repeat analyses except YE886, which was incompletely digested on several occasions. Thus, the reproducibility of the REAC patterns was 98.6%. To further investigate the reproducibility, seven subcultures of the same strain (PA11400) were set up on different days. Each subculture was then processed separately by the method described above and subjected to electrophoresis in the same gel. No significant differences between the seven REAC patterns could be detected. Comparison of genotypic and phenotypic typing methods. The discriminatory power of a typing method is its ability to distinguish between unrelated strains. It is determined by the number of types defined by the method in question and the relative frequency of these types (9). Simpson's index of diversity, which presents these two facets of discrimination as a single numerical value, was used to compare REAC with the other methods employed (Table 2). Although the isolates examined were not a random selection or representative of the wide diversity encountered within Y. enterocolitica, this index allows objective assessment of discriminatory power within the population of test isolates which included the most important pathogenic serogroups. Since serogroup affiliation was used as the basis for preselection of the isolates, 0-antigen typing could not be compared with the other methods. Of the six methods compared, antimicrobial susceptibility typing proved to have the greatest overall discriminatory power, followed by REAC, H-antigen typing, REAP, phage typing, and biotyping (in decreasing order of discrimination) (Table 2). Comparison of human and porcine isolates. Part of the study involved 23 isolates from pigs and pork, all belonging to serogroups associated with disease in humans. No differences in REAC patterns between porcine isolates and human clinical isolates belonging to serogroups 03, 08, or 09 could be demonstrated (Table 1). In order to investigate the phenotypic and genotypic diversity among strains from the same country, 13 isolates of serogroup 03, biovar 4, phagevar VIII from human patients and pigs in Norway were included. Although these strains could not be distinguished from each other by REAC, considerable variations in Hantigen pattern and antibiotic resistance were found, and one strain also exhibited a deviating REAP profile (Table 1). DISCUSSION Restriction fragment patterns of chromosomal DNA or total DNA preparations are difficult to compare because of the large number of bands produced. We achieved good
separation of the heaviest fragments by using the four-base cutter HaeIII and electrophoresis on thin polyacrylamide gels. Results were easy to interpret and reproducible. Comparison of the discriminatory power of six phenotypic and genotypic typing methods revealed that REAC ranked second, above H-antigen typing but below antimicrobial susceptibility testing. However, these results are valid only for the selection of isolates included in this study and are not necessarily representative of the wide diversity of strains found within the whole species. An advantage of typing methods based on the investigation of genotype is that the same set of reagents and equipment can be used (only small modifications are required) for many different bacteria (15, 26). In contrast, phage typing requires a battery of phages and indicator strains and is standardized for only a few species of bacteria. Serotyping likewise demands a large number of antisera which are difficult and expensive to prepare and standardize. Another advantage of genotypic methods is that they avoid problems associated with gene expression in vitro (15, 26). Many phenotypic characteristics vary depending on certain environmental or culture-related conditions, a circumstance which gives rise to diverging results between different laboratories. Of the two genotypic methods employed, REAP was the easiest to perform. Moreover, the results obtained by this method were easier to interpret than the complicated DNA fragment patterns obtained by REAC. However, in this study, a considerably better discrimination between isolates was achieved with REAC than with REAP, although the latter method involved the use of two restriction enzymes while REAC was based on the use of only one. Another advantage of REAC is that the method can be applied even if the bacterium to be studied has accidentally lost the virulence plasmid, while REAP, obviously, can only be employed if the plasmid is present. However, since presence of the virulence plasmid has a significant effect on the REAC patterns obtained (Fig. 1), one should ensure that all strains being compared are either plasmid cured or plasmid bearing. REAC and REAP both permitted differentiation of isolates which were indistinguishable by the phenotypic methods employed. On the other hand, it was in many cases possible to achieve a finer degree of differentiation of isolates with the same REAC or REAP patterns by using one or more of the phenotypic methods (Table 1). As regards Y. enterocolitica, geographic origin, ecologic characteristics, and pathogenic significance are closely correlated with certain combinations of serogroup, biovar, and phagevar (16). These classical phenotypic parameters thus provide valuable information on
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the strains, even though they should be combined with other methods such as REAC, REAP, or H-antigen typing if optimal differentiation is to be achieved. The discriminatory power of the genotypic methods was greatest within serogroup 08 (Table 2). Since this serogroup is homogeneous with regard to biovar and phagevar, there has long been a need for methods which permit a finer degree of differentiation in epidemiological investigations (3). A new bacteriophage typing system which allows greater differentiation of 08 strains has been described (3). The results of our investigation show that restriction enzyme analysis presents itself as an effective alternative or supplement to traditional methods, including phage typing, for the analysis of serogroup 08. Most isolates of Y. enterocolitica possess H antigens consisting of several subfactors. In a previous study, 117 serovars within Y. enterocolitica were defined on the basis of H-antigen patterns. The H antigens are monophasic and remain stable after repeated subcultivation and after storage as stab cultures (2). The great variability in H antigens demonstrated in the present and previous studies allowed differentiation of strains which could not be distinguished by any of the other typing methods employed. Even though the preparation and standardization of specific antisera are both time-consuming and costly, H-antigen typing thus nevertheless is a valuable supplement to 0-antigen typing and biochemical characterization of Y. enterocolitica in epidemiological investigations. Antimicrobial susceptibility testing is a highly standardized method employed in a large number of laboratories. However, susceptibility patterns may be influenced by local antibiotic usage, which may make differentiation of outbreak strains from epidemiologically unrelated strains difficult. Of the six typing methods which were compared in this study, antimicrobial susceptibility testing showed the greatest variability in all the serogroups investigated (Tables 1 and 2). However, on the basis of the investigation of strains from three outbreaks, Baker and Farmer (3) concluded that even though antimicrobial susceptibility testing contributed to the differentiation of the isolates, results were less reliable than those obtained by serotyping and phage typing. The differences in antibiograms reported in the present study should be interpreted with caution. Although the definition of resistance versus susceptibility is based on significant differences in zone sizes, a number of isolates showed intermediate values, indicating that the antibiograms are not necessarily completely reproducible. Three strains of serogroup 08 included in this study were originally isolated in connection with an outbreak of foodborne yersiniosis in a summer camp in Sullivan County, New York, in 1981 (strains 1700, 1821, and 1824) (M. Shayegani, personal communication). It is interesting that while all strains had the same REAC and REAP profiles, one strain exhibited a deviating H-antigen pattern and a slightly different antibiogram, compared with the other two (Table 1).
In a parallel study, we employed multilocus enzyme electrophoresis to investigate allelic variations in the chromosomal genomes of the same strains included in this study (7). Results revealed only slight variability between strains of the same serogroup. The method would have given an overall discrimination index of 0.752, indicating it to be less suitable for tracing epidemiologically related Y. enterocolitica strains. From an epidemiological point of view, it is interesting that isolates from pigs showed the same REAC and REAP
J. CLIN. MICROBIOL.
patterns as human isolates, regardless of the serogroup affiliation. The same H antigens, biovars, phagevars, and antibiograms were also represented among isolates from both humans and pigs. Even though the relative frequency of different variants in pigs and humans was not systematically investigated, these observations nevertheless reinforce our supposition that pigs constitute an important source of human infection. ACKNOWLEDGMENTS The antibiotic susceptibility testing was kindly performed by A. Katz. We thank Bill Davies and Henning S0rum for helpful discussions. The technical assistance of Traute Vardund, Kari Dommarsnes, and Berit Gamnes is gratefully acknowledged. This work was supported by grants from the Norwegian Council for Agricultural Research. LITERATURE CITED 1. 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 the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species. Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 261:299-310. 3. Baker, P. M., and J. J. Farmer III. 1982. New bacteriophage typing system for Yersinia enterocolitica, Yersinia kristensenii, Yersiniafrederiksenii, and Yersinia intermedia: correlation with serotyping, biotyping, and antibiotic susceptibility. J. Clin. Microbiol. 15:491-502. 4. Balows, A. 1976. Performance standards for antimicrobial disc susceptibility tests as used in clinical laboratories, p. 138-155. In A. Balows (ed.), Current techniques for antibiotic susceptibility testing. Charles C Thomas, Publisher, Springfield, III. 5. Bjorvatn, B., and B.-E. Kristiansen. 1985. Molecular epidemiology of bacterial infections. Clin. Lab. Med. 5:437-445. 6. Bradbury, W. C., A. D. Pearson, M. A. Marko, R. V. Congi, and J. L. Penner. 1984. Investigation of a Campylobacter jejuni outbreak by serotyping and chromosomal restriction endonuclease analysis. J. Clin. Microbiol. 19:342-346. 7. Caugant, D. A., S. Aleksic, H. H. Mollaret, R. K. Selander, and G. Kapperud. 1989. Clonal diversity and relationships among strains of Yersinia enterocolitica. J. Clin. Microbiol. 27:26782683. 8. Cornelis, G. 1981. Antibiotic resistance in Yersinia enterocolitica, p. 55-71. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 9. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466. 10. Hurvell, B. 1981. Zoonotic Yersinia enterocolitica infection: host range, clinical manifestations, and transmission between animals and man, p. 145-160. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 11. Kaper, J. B., H. B. Bradford, N. C. Roberts, and S. Falkow. 1982. Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast. J. Clin. Microbiol. 16:129-134. 12. Kapperud, G. 1981. Survey on the reservoirs of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in Scandinavia. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 89:29-35. 13. Kristiansen, B.-E., B. S0rensen, B. Bjorvatn, E. S. Falk, E. Fosse, K. Bryn, L. O. Fr0holm, P. Gaustad, and K. B0vre. 1986. An outbreak of group B meningococcal disease: tracing the causative strain of Neisseria meningitidis by DNA fingerprinting. J. Clin. Microbiol. 23:764-767. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, p. 368-369. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Mayer, L. W. 1988. Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance. Clin. Microbiol. Rev. 1:228-243.
VOL. 28, 1990 16. Mollaret, H. H., H. Bercovier, and J. A. Alonso. 1979. Summary of the data received at the WHO Reference Center for Yersinia enterocolitica. Contrib. Microbiol. Immunol. 5:174-184. 17. Moliaret, H. H., and P. Nicolle. 1965. Sur la fréquence de la lysogénie dans l'espèce nouvelle Yersinia enterocolitica. C.R. Acad. Sci. 260:1027-1029. 18. Nesbakken, T., B. Gondrosen, and G. Kapperud. 1985. Investigation of Yersinia enterocolitica, Yersinia enterocolitica-like bacteria, and thermotolerant campylobacters in Norwegian pork products. Int. J. Food Microbiol. 1:311-320. 19. Nesbakken, T., G. Kapperud, H. S0rum, and K. Dommarsnes. 1987. Structural variability of 40-50 Mdal virulence plasmids from Yersinia enterocolitica. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 95:167-173. 20. Nicoile, P. 1973. Yersinia enterocolitica, p. 377-387. In H. Rische (ed.), Lysotypie und andere spezielle epidemiologische Laboratoriums-methoden. VEB Gustav Fisher Verlag, Jena, German Democratic Republic. 21. Perry, R. D., and R. R. Brubaker. 1983. Vwa+ phenotype of Yersinia enterocolitica. Infect. Immun. 40:166-171. 22. Pulkkinen, L., I. Granberg, K. Granfors, and A. Toivanen. 1986. Restriction map of virulence plasmid in Yersinia enterocolitica 0:3. Plasmid 16:225-227.
REAC AND REAP OF YERSINIA SPP.
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23. Tauxe, R. V., J. Vandepitte, G. Wauters, S. M. Martin, V. Goossens, P. De Mol, R. Van Noyen, and G. Thiers. 1987. Yersinia enterocolitica infections and pork: the missing link. Lancet i:1129-1132. 24. Tenover, F. C. 1988. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbiol. Rev. 1:82-101. 25. Tompkins, L. S., N. J. Troup, T. Woods, W. Bibb, and R. M. McKinney. 1987. Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis. J. Clin. Microbiol. 25:1875-1880. 26. Wachsmuth, K. 1985. Genotypic approaches to the diagnosis of bacterial infections: plasmid analyses and gene probes. Infect. Control 6:100-109. 27. Wauters, G. 1981. Antigens of Yersinia enterocolitica, p. 41-53. In E. J. Bottone (ed.), Yersinia enterocolitica. CRC Press, Inc., Boca Raton, Fla. 28. Wauters, G., M. Janssens, A. G. Steigerwalt, and D. J. Brenner. 1988. Yersinia mollaretii sp. nov. and Yersinia bercovieri sp. nov., formerly called Yersinia enterocolitica biogroups 3A and 3B. Int. J. Syst. Bacteriol. 38:424-429. 29. Wauters, G., K. Kandolo, and M. Janssens. 1987. Revised biogrouping scheme of Yersinia enterocolitica. Contrib. Microbiol. Immunol. 9:14-21.
