JOURNAL OF CLINICAL MICROBIOLOGY, June 1996, p. 1535–1539 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 6
Subspecies Typing of Vibrio parahaemolyticus by Pulsed-Field Gel Electrophoresis HIN-CHUNG WONG,1* KANG-TZU LU,1 TZE-MING PAN,2 CHI-LUNG LEE,2 3 AND DANIEL YANG-CHI SHIH Department of Microbiology, Soochow University, Taipei, Taiwan 11102,1 and Bacteriology Division, National Institute of Preventive Medicine,2 and Food Microbiology Division, National Laboratories of Food and Drug,3 Taipei, Taiwan 11513, Republic of China Received 2 November 1995/Returned for modification 9 January 1996/Accepted 28 February 1996
Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other coastal regions. We report on the development of a pulsed-field gel electrophoresis (PFGE) method for the molecular typing of this pathogen. Genomic DNA was digested with SfiI, and the fragments were resolved on 1% agarose with a contour-clamped homogeneous electric field apparatus set at 190 V and a pulse time of 3 to 80 s. A total of 130 selected isolates obtained from outbreaks during 1993 and 1994 on Taiwan were also characterized by this PFGE method. These isolates were grouped into 14 PFGE types which consisted of one to six patterns, and a total of 39 patterns were identified. Most of these domestic clinical isolates could be clustered into several major types (types A, B, C, and G). These major types showed relatively low degrees of similarity to several foreign strains and other domestic but environmental strains. Strain CCRC12863, which originated from Japan, was close to the group consisting of F, G, and H PFGE types, suggesting a clonal relationship between this Japanese strain and other domestic isolates. Vibrio parahaemolyticus is a halophilic gram-negative bacterium that causes acute gastroenteritis in humans. It is one of the most important food-borne pathogens in Taiwan, Japan, and other coastal regions (4). Most clinical isolates of this pathogen are hemolytic on Wagatsuma agar (Kanagawa positive). In contrast, most environmental isolates are Kanagawa negative (5). Isolates of V. parahaemolyticus can be differentiated by serotypes. Commercial serotyping antisera are produced in Japan (Denka Seiken, Tokyo, Japan) and could be available in other countries. At present, 11 O groups and 71 K types are identified by these commercial antisera. Usually, the serotyping method cannot differentiate among all isolates originating from different regions. In practice, the determination of K antigens in V. parahaemolyticus can be interfered with by the presence of certain O antigens because of the use of OK type antisera, and O type agglutination can also be inhibited by K antigens (reference no. 1433; Denka Seiken). For epidemiological investigations, dependable molecular methods need to be developed so that all strains can be typed. Several molecular methods have been developed for the typing of Vibrio species which are important pathogens in humans and aquacultured animals, for example, ribotyping and pulsed-field gel electrophoresis (PFGE) for V. cholerae (1), PFGE for V. vulnificus (3), and ribotyping and PFGE for V. anguillarum (7). However, molecular typing methods for the subspecies differentiation of V. parahaemolyticus have not been published. Here we report on the development of PFGE for the molecular typing of V. parahaemolyticus, and we also characterized by this procedure selected clinical isolates obtained during 1993 and 1994 on Taiwan.
MATERIALS AND METHODS Bacterial strains. One hundred thirty clinical isolates were selected from cultures of stool samples collected from individuals involved in food poisoning outbreaks during 1993 and 1994 on Taiwan, and the isolates were subjected to PFGE analysis (Table 1). These isolates were identified by API 20E identification strips (API system; Analytab Products, Montalieu-Vercieu, France) and also by a conventional method (8). Three strains, CCRC12863 (ATCC 17803), CCRC12864 (ATCC 27519), and CCRC12866, which originated from foreign countries, plus three local environmental isolates (CCRC12963, S-2, and CHAO) were also examined (Table 1). Cultures of these isolates were stored at 2858C in tryptic soy broth (Difco Laboratories, Detroit, Mich.)–3% NaCl containing 20% glycerol. DNA extraction and digestion for PFGE. Bacteria on tryptic soy agar (Difco)–3% NaCl were transferred to 5 ml of tryptic soy broth (Difco)–3% NaCl in a 50-ml plastic centrifuge tube and were cultured overnight at 378C with shaking at 160 rpm. Bacterial cells were harvested by centrifugation and were resuspended in 2 ml of a buffer containing 10 mM Tris, 100 mM EDTA, and 1 mM NaCl (pH 8.0). Agarose plugs were prepared by mixing equal volumes of bacterial suspensions with 1.5% low-melting-point agarose (FMC Corp., Rockland, Maine). The bacterial cells in the agarose plugs were lysed by treatment with lysis solution containing 1 mg of lysozyme per ml and 0.1% N-sodium lauroyl sarcosine at 378C for 24 h and were then treated with proteinase K (0.5 mg/ml in 0.5 M EDTA and 1% N-sodium lauroyl sarcosine) at 458C for 48 h and washed three times (for 30 min each time) with TE buffer (10 mM Tris z HCl, 1 mM EDTA). One section of the plug (4 by 9 by 1.2 mm) was equilibrated with enzyme buffer and was placed in 100 ml of fresh buffer containing 10 U of SfiI (New England BioLabs, Beverly, Mass.), and the mixture was incubated at 48C for 16 h and was then incubated at 378C for 48 h. PFGE. High-molecular-weight restriction fragments were resolved on a 1% agarose gel in 0.53 Tris-borate-EDTA (TBE) buffer by using a contour-clamped homogeneous electric field (CHEF) apparatus (CHEF-DR II; Bio-Rad Laboratories, Richmond, Calif.) (7). The running conditions were 190 V for 22.4 h at 148C, with pulse times of 3 to 80 s. To separate unclarified fragments of less than 582 kb, PFGE was also performed at pulse times of 1 to 40 s. Bacteriophage lambda DNA ladder PFGE markers (New England Biolabs) were used as molecular size markers. After electrophoresis, the gels were stained with ethidium bromide (Sigma Co., St. Louis, Mo.), destained in distilled water, and photographed with a UV transilluminator (Flou-Link 312; Vilber Lourmat, Torey, France). Similarities among patterns. The size of each band was determined with Stratascan Analysis software (Stratagene, La Jolla, Calif.), and line representations of the gels were produced by the software Microsoft Excel, version 5.0, for Windows, but for purposes of numerical analysis, bands of from 20 to 690 kb were grouped in 10-kb ranges. Bands larger than 690 kb were grouped into two ranges, 690 to 776 kb and those larger than 776 kb (2). Data were coded as 0 (negative) or 1 (positive). The hierarchical cluster analysis was done by the average linkage method with the squared Euclidean distance measure. A den-
* Corresponding author. Mailing address: Department of Microbiology, Soochow University, Taipei, Taiwan 11102, Republic of China. Phone: (886) 2-8819471, extension 6852. Fax: (886) 2-8831193. Electronic mail address:
[email protected]. 1535
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TABLE 1. V. parahaemolyticus isolate types by PFGEa
TABLE 1—Continued
PFGE type
Strain no.
Serotype
TDH
Location
Outbreak no.
A1
153 322 351 353 355 358 363 392
ND ND ND ND ND ND ND K-8
1 1 1 1 1 1 1 1
Taipei Miao-Li Taipei Taipei Taipei Taipei Kaoshung Taipei
2 9 10 10 10 10 11 13
A2
399
K-12
1
Kaoshung
14
A3
536
ND
1
Kaoshung
19
B1
302 304
K-29 K-29
1 1
Miao-Li Miao-Li
7 7
B2
305 324 425
K-29 ND K-29
1 1 1
Miao-Li Miao-Li Chia-I
7 9 15
B3
314 416 420
K-29 K-29 K-29
1 2 2
Miao-Li Chia-I Chia-I
7 15 15
B4
414
ND
1
Chia-I
15
B5
640 641
K-29 K-29
1 1
Kaoshung Kaoshung
34 34
B6
155
ND
1
Taipei
2
C1
075 171
ND ND
1 1
I-Lan Kaoshung
1 3
C2
481 489 492 638 639 647 669
K-15 K-15 K-15 K-15 K-15 K-5 K-56
1 1 1 1 1 1 1
Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Chia-I Chia-I
17 17 17 33 33 35 39
C3
636
K-15
1
Kaoshung
32
C4
677 670
K-8 K-56
1 2
Taipei Chia-I
41 39
C5
675 676 679 680 682 684 687
K-8 K-8 K-8 K-8 ND ND ND
1 1 1 1 1 1 1
Taipei Taipei Peng-Hu Peng-Hu Peng-Hu Peng-Hu Peng-hu
41 41 42 42 42 42 42
364 396 488 527 617 618 619 620 621 622 623 624
ND K-12 K-15 K-10 ND ND ND K-15 ND K-15 ND ND
1 2 1 1 1 1 1 1 1 1 1 1
Kaoshung Taipei Kaoshung Tainan Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung
11 13 17 18 29 29 29 29 29 29 30 30
C6
Continued
PFGE type
Strain no.
Serotype
TDH
Location
Outbreak no.
625 626 627 629 630 631 632 633 634 635 637 702
K-15 ND ND ND ND K-15 K-15 K-15 K-15 K-15 K-15 ND
1 1 1 1 1 1 1 1 1 1 1 1
Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung
30 31 31 31 31 31 32 32 32 32 32 45
D1
576 588 599 589 590
K-3 K-3 ND K-3 K-3
2 1 1 2 1
Tainan Tainan Kaoshung Tainan Tainan
24 24 26 24 24
D2
283 284
ND ND
1 1
Kaoshung Kaoshung
5 5
E1
579 584 597 609 614
K-3 K-3 ND ND ND
1 1 2 1 2
Tainan Tainan Tainan Hsin-chu Kaoshung
24 24 25 27 28
E2
613
ND
2
Kaoshung
28
E3
615
ND
2
Kaoshung
28
F1
642 643 644 645 646 648 649
K-5 K-5 K-5 K-5 K-5 K-5 K-5
1 1 1 1 1 1 1
Chia-I Chia-I Chia-I Chia-I Chia-I Chia-I Chia-I
35 35 35 35 35 35 35
F2
650 671
ND K-60
1 1
Taichung Hua-Lien
36 40
G1
381
ND
1
Taipei
12
G2
430 556 681
K-60 K-29 K-8
1 1 1
Yun-Lin Taipei Peng-Hu
16 23 42
G3
435
K-60
1
Yun-Lin
16
G4
664 665 666 667 672 673
ND K-56 K-56 K-56 K-60 K-60
1 1 1 1 1 1
Chia-I Chia-I Chia-I Chia-I Hua-Lien Hua-Lien
39 39 39 39 40 40
H1
548 701
K-63 K-15
1 1
Chi-Lung Kaoshung
20 44
I1
616
K-15
1
Kaoshung
29
I2
628
ND
1
Kaoshung
31
J1
541
K-10
1
Kaoshung
19
J2
651
K-56
2
Kaoshung
37
Continued on following page
MOLECULAR TYPING OF V. PARAHAEMOLYTICUS
VOL. 34, 1996
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TABLE 1—Continued PFGE type
Strain no.
Location
Outbreak no.
Serotype
TDH
652 653 654 655 656 657 658 659 660 661
K-56 K-56 K-56 ND K-56 K-56 K-56 ND ND ND
2 2 2 2 2 2 2 2 2 2
Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung Kaoshung
37 37 37 37 37 38 38 38 38 38
K
198
K-57
1
Kaoshung
4
L1
292
ND
2
Tainan
6
L2
736 740
K-8 ND
1 1
Ping-Tung Ping-Tung
47 48
M1
318 668
K-12 K-56
1 1
Chia-I Chia-I
8 39
M2
424
K-29
2
Chia-I
15
M3
663 691 729 731 732
K-56 K-41 K-41 K-41 K-41
2 1 1 1 1
Chia-I Hua-Lian Tao-Yuan Tao-Yuan Tao-Yuan
39 43 46 46 46
N
550 553 CCRC12863 CCRC12864 CCRC12866
K-4 K-4 ND K30 ND
1 1 1 1 1
21 22
CCRC12963
K30
2
S-2
ND
2
CHAO
ND
2
Taipei Taichung Japan (clinical) Louisiana (clinical) New Zealand (from a clam) Taiwan (from a shrimp) Laboratory stock (environmental) Laboratory stock (environmental)
a TDH, thermostable direct hemolysin; ND, not determined; CCRC, Culture Collection and Research Center, Hsin-Chu, Taiwan. Strains CCRC12863 and CCRC12864 originated from the American Type Culture Collection (ATCC), Rockville, Md., and are designated ATCC 17803 and ATCC 27519, respectively.
drogram was produced with the SPSS program for Windows, release 6.0 (SPSS Inc., Chicago, Ill.) (6). Strains with dissimilarity values larger than 12 were arbitrarily grouped into different PFGE types.
RESULTS AND DISCUSSION Genomic DNA of V. parahaemolyticus CCRC12864 was digested with 16 restriction enzymes, namely, AccI, AseI, AvaI, BamHI, BstXI, ClaI, EcoRI, HindIII, NdeI, KpnI, PstI, SmaI, SfiI, XbaI, XhoI, and XhoII, and was analyzed by PFGE. Most of these enzyme-restricted fragments were smaller than 145 kb; the exceptions were BamHI and SfiI. BamHI digestion gave unresolved bands larger than 600 kb, while SfiI digestion gave 17 clear and discernible bands in the range of 48 to 727 kb. Several other selected strains (strains 075, 153, 155, and 171) were later digested with SfiI and gave a suitable distribution of bands. Therefore, SfiI was chosen for the PFGE analysis of all strains described below.
FIG. 1. PFGE of some selected V. parahaemolyticus isolates. The conditions for PFGE were as follows: 1% agarose gel, 0.53 TBE buffer, 190 V, pulse times of 3 to 80 s, and a run time of 22.4 h. Lanes: 1, isolate 618; 2, isolate 617; 3, isolate 615; 4, isolate 614; 5, isolate 613; 6, isolate 657; 7, isolate 642; 8, isolate 590; 9, bacteriophage lambda DNA ladder as a PFGE marker (in kilobases).
From 1993 to 1995, more than 300 isolates of V. parahaemolyticus were collected from individuals involved in food poisoning outbreaks in different regions of Taiwan. In the present study, 130 isolates were selected from among the isolates collected over the first 2 years and were subjected to PFGE analysis. When the pulse time was set at 1 to 40 s, restricted fragments smaller than 582 kb were clearly resolved. When the pulse time was set at 3 to 80 s, fragments larger than 582 kb could also be resolved, and the latter condition was recommended for use in the PFGE analysis of all V. parahaemolyticus isolates (Fig. 1). By using these conditions, 12 to 18 SfiI-restricted fragments were identified from these isolates (Fig. 2). After hierarchical cluster analysis, these 130 isolates could be grouped into 14 PFGE types (types A to N), and each type was subdivided into one to six patterns. A total of 39 PFGE patterns were identified (Fig. 3; Table 1). According to their similarities, these patterns were clustered into several groups. The major groups consisted of types A, B, C, and G. The type K isolate, from only one outbreak, had a low degree of similarity to the majority of the domestic isolates (Fig. 3). These PFGE patterns were not related to the serotypes, while PFGE was more discriminative than serotyping. Most of the serotypes could be grouped into several PFGE patterns; for example, serotype K56 from different outbreaks could be grouped into PFGE patterns C2, C4, G4, J2, K, M1, and M3. Isolates from many of the outbreaks could be differentiated into several PFGE patterns, for example, B2, B3, B4, and M2 in outbreak 15 (Table 1). The PFGE patterns were also not related to the Kanagawa phenomenon. Thermostable direct hemolysin-negative isolates occurred among isolates with PFGE patterns B, C, D, E, M, and J (Table 1). Some of these PFGE types more frequently originated from certain regions of Taiwan; for example, types C, I, J, K, and L were from southern Taiwan (Kaoshung, Tainan, Ping-Tung, and Peng-Hu) (Table 1). It is unlikely, however, that the PFGE types are specific to particular locales on this island, since the translocation of consumers and commodities is very intense on Taiwan, enabling the rapid and widespread dispersion of microorganisms. On the other hand, many other PFGE types and patterns were recovered from several locations on Taiwan (Table 1). Nevertheless, most of these domestic clinical isolates could be clustered into major groups, and these groups showed low degrees of similarity to foreign strains (strain CCRC12864
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FIG. 2. PFGE types and patterns of the clinical V. parahaemolyticus isolates obtained on Taiwan. A1 to N, different PFGE types and patterns; a, strain S-2; b, strain CHAO; c, CCRC12863; d, CCRC12864; e, CCRC12866; f, CCRC12963; l, bacteriophage lambda DNA ladder as a PFGE marker (in kilobases).
from the United States and strain CCRC12866 from The Netherlands) or to the other three domestic environmental strains (strains S2, CHAO, and CCRC12963) (Fig. 3). Strain CCRC12863, which originated from Japan, was close to the group consisting of PFGE types F, G, and H, suggesting a clonal relationship between this Japanese strain and other domestic isolates. These PFGE patterns seem to be specific for domestic clinical isolates or for strains originating from an adjacent geographical area. More foreign strains should be analyzed to confirm the specificities of these PFGE patterns for V. parahaemolyticus. In other studies, PFGE subdivided the V. anguillarum collection into 35 groups, and strains with origins in different countries were shown to have different PFGE types (7). PFGE of V. cholerae O1 could also identify strains of different geographic origins (1). In conclusion, we have reported on a PFGE procedure for the molecular typing of V. parahaemolyticus. A total of 130 domestic clinical isolates were grouped into 14 PFGE types and 39 patterns, and most of the environmental isolates and foreign strains were not similar to the majority of the domestic clinical isolates examined.
ACKNOWLEDGMENT This research was supported by Department of Health, Republic of China (grant DOH84-FS-003).
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FIG. 3. Dendrogram showing the clustering of PFGE patterns (SfiI) for the V. parahaemolyticus isolates and strains listed in Table 1. The dendrogram was based on the squared Euclidean distance measure and average linkage clustering method by the SPSS program for Windows, release 6.0. The dissimilarity units are arbitrary, being based on the squared Euclidian distance measure. Strains with dissimilarity values larger than 12 were arbitrarily grouped into different PFGE types. Letters in the left column designate the PFGE pattern or the strain number. Strains designated with five numbers were from cultures of the Culture Collection and Research Center (CCRC; Hsin-Chu, Taiwan). Strains S-2 and CHAO were identical.
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