Virulence Profiles of Vibrio vulnificus in German ...

Supplementary Information

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates Table S1. Sampling sites, classification and bathing water quality. Geographical Region Sampling Site No. 1 2 3 4 5 North Sea 6 7 8 9

Baltic Sea

10 11 12 13 14 15 16 17 18 19 20

Sampling Site No. of Strains Classification (Coastal Waters) a Bathing Water Quality b Borkum 1 euhaline open coastal waters excellent Dyksterhusen 3 mesohaline inner coastal waters good Jemgum 1 mesohaline inner coastal waters no designated beach Burhave 8 mesohaline inner coastal waters excellent Dedesdorf 9 mesohaline inner coastal waters no designated beach Kleinensiel 2 mesohaline inner coastal waters no designated beach Bremerhaven 16 mesohaline inner coastal waters no designated beach Wremen 5 mesohaline inner coastal waters excellent Altenbruch 3 mesohaline inner coastal waters excellent k.A. 2 Mönkeberg 1 mesohaline inner coastal waters Excellent Kiel-Dietrichsdorf 1 mesohaline inner coastal waters no designated beach Wohlenberger Wiek 2 mesohaline inner coastal waters good Kühlungsborn 1 mesohaline open coastal waters excellent Warnemünde 6 mesohaline open coastal waters excellent Darss-Zingster Bodden chain, station 9 2 mesohaline inner coastal waters no designated beach Darss-Zingster Bodden chain, station 8 2 mesohaline inner coastal waters no designated beach Darss-Zingster Bodden chain, station 7 4 mesohaline inner coastal waters no designated beach Greifswalder Bodden, station 5 2 mesohaline inner coastal waters no designated beach Greifswalder Bodden, station 4 3 mesohaline inner coastal waters no designated beach Greifswalder Bodden, station 3 5 mesohaline inner coastal waters no designated beach

Int. J. Environ. Res. Public Health 2015, 12

S2 Table S1. Cont.

Geographical Region Sampling Site No. Sampling Site No. of Strains Classification (Coastal Waters) a Bathing Water Quality b 21 Greifswalder Bodden, station 2 4 mesohaline inner coastal waters no designated beach 22 Lubmin 4 mesohaline inner coastal waters excellent 23 Karlshagen 3 mesohaline open coastal waters excellent 24 Trassenheide 1 mesohaline open coastal waters excellent Baltic Sea 25 Greifswalder Bodden, station 1 2 mesohaline inner coastal waters no designated beach 26 Binz 7 mesohaline open coastal waters excellent 27 Rügen 1 mesohaline inner coastal waters no designated beach a

according to the European Water Framework Directive 2000/60/EC. results from 2011.

b

according to the requirements of the European Bathing Water Directive 2006/7/EC;

Table S2. Detailed sampling information of V. vulnificus isolates examined in this study. Strain ID Origin Sampling Site No. a Sampling Site Name Seawater Temperature (°C) Seawater Salinity (psu) Sampling Date VN-0279 B-sw 12 Wohlenberger Wiek 15.5 13 06.09.2011 VN-2813 N-sw n.s. n.s. n.s. 16.05.2011 VN-2814 N-sw n.s. n.s. n.s. 16.05.2011 VN-2961 B-sw 11 Kiel-Dietrichsdorf 18 n.s. 25.07.2011 VN-2969 B-sw 10 Mönkeberg 17.4 n.s. 25.07.2011 VN-3363 N-sd 4 Burhave 25.6 21.7 12.07.2010 VN-3364 N-sd 4 Burhave 25.6 21.7 12.07.2010 VN-3366 N-sd 4 Burhave 25.6 21.7 12.07.2010 VN-3367 N-sw 7 Bremerhaven 24.8 17.0 12.07.2010 VN-3368 N-sw 7 Bremerhaven 24.8 17.0 12.07.2010 VN-3369 N-sw 7 Bremerhaven 24.8 17.0 12.07.2010 VN-3373 N-sd 5 Dedesdorf 20.4 8.2 12.07.2010 VN-3374 N-sd 5 Dedesdorf 20.4 8.2 12.07.2010 VN-3378 N-sw 2 Dyksterhusen 20.4 20.5 20.07.2010


S3 Table S2. Cont.

Strain ID Origin Sampling Site No. a Sampling Site Name Seawater Temperature (°C) Seawater Salinity (psu) Sampling Date VN-3379 N-sw 2 Dyksterhusen 20.4 20.5 20.07.2010 VN-3394 N-sd 8 Wremen 21.7 22.5 10.08.2010 VN-3398 N-sw 8 Wremen 21.7 22.5 10.08.2010 VN-3403 N-sw 8 Wremen 21.7 22.5 10.08.2010 VN-3408 N-sw 2 Dyksterhusen 19 14.2 17.08.2010 VN-3410 N-sw 4 Burhave 18.7 25.1 18.08.2010 VN-3411 N-sd 4 Burhave 18.7 25.1 18.08.2010 VN-3412 N-sw 7 Bremerhaven 20.5 19.3 18.08.2010 VN-3415 N-sw 7 Bremerhaven 20.5 19.3 18.08.2010 VN-3418 N-sd 7 Bremerhaven 20.5 19.3 18.08.2010 VN-3419 N-sw 5 Dedesdorf 20.7 6.2 18.08.2010 VN-3426 N-sd 5 Dedesdorf 20.7 6.2 18.08.2010 VN-3442 N-sd 1 Borkum 16 29.8 24.08.2010 VN-3443 N-sd 4 Burhave 15 11.8 07.09.2010 VN-3444 N-sd 4 Burhave 15 11.8 07.09.2010 VN-3446 N-sd 7 Bremerhaven 15 11.8 07.09.2010 VN-3448 N-sw 7 Bremerhaven 17.5 14.6 07.09.2010 VN-3451 N-sw 7 Bremerhaven 17.5 14.6 07.09.2010 VN-3454 N-sd 7 Bremerhaven 17.5 14.6 07.09.2010 VN-3457 N-sd 7 Bremerhaven 17.5 14.6 07.09.2010 VN-3461 N-sw 5 Dedesdorf 17.7 1.9 07.09.2010 VN-3465 N-sd 5 Dedesdorf 17.7 1.9 07.09.2010 VN-3467 N-sd 5 Dedesdorf 17.7 1.9 07.09.2010 VN-3477 N-sw 7 Bremerhaven 14.1 14.9 05.10.2010 VN-3478 N-sd 7 Bremerhaven 14.1 14.9 05.10.2010 VN-3479 N-sd 5 Dedesdorf 15.6 5.3 05.10.2010


S4 Table S2. Cont.

Strain ID VN-3494 VN-3496 VN-3498 VN-3500 VN-3506 VN-3518 VN-3529 VN-3533 VN-3536 VN-3538 VN-3539 VN-3541 VN-3542

Origin N-sw N-sw N-sd N-sd N-sw N-sw N-sw N-sw N-sw N-sw N-sw N-sw N-sw

Sampling Site No. a 7 7 4 7 5 9 6 8 9 6 3 8 9

VN-3904

B-sd

19

VN-3905

B-sd

19

VN-3906

B-sd

18

VN-3909

B-sd

17

VN-3910

B-sd

17

VN-3912

B-sd

16

Sampling Site Name Bremerhaven Bremerhaven Burhave Bremerhaven Dedesdorf Altenbruch Kleinensiel Wremen Altenbruch Kleinensiel Jemgum Wremen Altenbruch Greifswalder Bodden, station 4 Greifswalder Bodden, station 4 Greifswalder Bodden, station 5 Darss-Zingster Bodden chain, station 7 Darss-Zingster Bodden chain, station 7 Darss-Zingster Bodden chain, station 8

Seawater Temperature (°C) 17.5 17.5 15 20.5 17.7 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Seawater Salinity (psu) 14.6 14.6 11.8 12.4 1.9 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Sampling Date 07.09.2010 07.09.2010 07.09.2010 11.07.2011 07.09.2010 12.06.2012 02.08.2012 07.08.2012 07.08.2012 13.08.2012 14.08.2012 21.08.2012 06.09.2012

19.0

6.6

05.07.2011

19.0

6.6

05.07.2011

19.3

7.2

05.07.2011

19.2

7.9

05.07.2011

19.2

7.9

05.07.2011

19.0

7.1

05.07.2011


VN-3914

B-sd

15

VN-3915

B-sd

15

VN-3919

B-sd

25

VN-3921

B-sd

21

VN-3922

B-sw

20

VN-3924

B-sd

20

VN-3925

B-sd

12

S5 Darss-Zingster Bodden chain, station 9 Darss-Zingster Bodden chain, station 9 Greifswalder Bodden, station 1 Greifswalder Bodden, station 2 Greifswalder Bodden, station 3 Greifswalder Bodden, station 3 Wohlenberger Wiek

18.4

7.3

05.07.2011

18.4

7.3

05.07.2011

18.4

6.7

06.07.2011

18.9

6.6

06.07.2011

19.1

6.4

06.07.2011

19.1

6.4

06.07.2011

18.9

10.7

01.08.2011


S6 Table S2. Cont.

Strain ID Origin VN-3926 VN-3927 VN-3928 VN-3929 VN-3931 VN-3932 VN-3934 VN-3935 VN-3937 VN-3946 VN-3947 VN-3948 VN-3959 VN-3960 VN-3961 VN-3962 VN-3964 VN-3965 VN-3966 VN-3968 VN-3969 VN-3970 VN-3971 VN-3972 VN-3973

B-sd B-sd B-sw B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw

Sampling Site No. a 25 21 20 20 20 19 17 16 16 21 21 18 22 23 23 22 26 26 26 26 26 22 24 23 13

Sampling Site Name Greifswalder Bodden, station 1 Greifswalder Bodden, station 2 Greifswalder Bodden, station 3 Greifswalder Bodden, station 3 Greifswalder Bodden, station 3 Greifswalder Bodden, station 4 Darss-Zingster Bodden chain, station 7 Darss-Zingster Bodden chain, station 7 Darss-Zingster Bodden chain, station 8 Greifswalder Bodden, station 2 Greifswalder Bodden, station 2 Greifswalder Bodden, station 5 Lubmin Karlshagen Karlshagen Lubmin Binz Binz Binz Binz Binz Lubmin Trassenheide Karlshagen Kühlungsborn

Seawater Temperature (°C) 18,2 19,5 18,9 18,9 18,9 19,7 19,7 19,7 20,7 13,4 13,4 12,6 26 21,1 21,1 20 n.s. n.s. n.s. n.s. n.s. n.s. 20,9 21,5 20,2

Seawater Salinity (psu) Sampling Date 7,3 6,2 6,2 5,9 5,9 6,3 5,6 5,6 3 6,7 6,7 7,6 6,5 5,4 5,4 6,4 6,7 6,7 6,7 6,7 6,7 6,3 6,6 6,5 8,3

02.08.2011 02.08.2011 02.08.2011 02.08.2011 02.08.2011 02.08.2011 03.08.2011 03.08.2011 03.08.2011 11.10.2011 11.10.2011 11.10.2011 05.07.2010 19.07.2010 19.07.2010 19.07.2010 23.07.2010 23.07.2010 23.07.2010 23.07.2010 23.07.2010 02.08.2010 02.08.2010 02.08.2010 02.08.2010


S7 Table S2. Cont.

Strain ID Origin Sampling Site No. a Sampling Site Name Seawater Temperature (°C) Seawater Salinity (psu) Sampling Date VN-3974 B-sw 14 Warnemünde 22,8 8 04.08.2010 VN-3975 B-sw 14 Warnemünde 22,8 8 04.08.2010 VN-3976 B-sw 14 Warnemünde 22,8 8 04.08.2010 VN-3977 B-sw 14 Warnemünde 22,8 8 04.08.2010 VN-3978 B-sw 14 Warnemünde 22,8 8 04.08.2010 VN-3979 B-sd 14 Warnemünde 22,8 8 04.08.2010 VN-3980 B-sw 26 Binz 21 6,6 09.08.2010 VN-3981 B-sw 22 Lubmin 5,4 19,5 11.07.2011 VN-3982 B-sw 26 Binz n.s. 6,7 23.07.2010 VN-5163 B-sw 27 Rügen n.s. n.s. 12.06.2011 N, North Sea; B, Baltic Sea; sw, seawater; sd, sediment; n.s., not specified. a Sampling site numbers shown in Figure 1.

Table S3. Allelic profiles of the 101 V. vulnificus isolates tested (new STs/alleles are displayed in red). Strain ID

MLST ST

MLST-Cluster

VN-0279 VN-2813 VN-2814 VN-2961 VN-2969 VN-3363 VN-3364 VN-3366 VN-3367 VN-3368 VN-3369

217 219 219 220 220 223 223 224 219 225 225

IIB I I IIB IIB I I I I IIA IIA

Clonal Complex (SLV-Level) Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton

Clonal Complex (TLV-Level) Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton

glp gyrB mdh metG purM dtdS lysA pntA pyrC tnaA 12 70 70 4 4 49 49 47 70 44 44

39 57 57 58 58 40 40 14 57 1 1

42 2 2 12 12 44 44 2 2 37 37

29 9 9 13 13 24 24 7 9 59 59

57 8 8 12 12 8 8 8 8 25 25

27 97 97 24 24 107 107 19 97 98 98

41 4 4 92 92 6 6 33 4 106 106

77 6 6 1 1 65 65 15 6 1 1

13 78 78 13 13 5 5 5 78 23 23

11 73 73 19 19 50 50 74 73 7 7


S8 Table S3. Cont.

Strain ID

MLST ST

MLST-Cluster

VN-3373 VN-3374 VN-3378 VN-3379 VN-3394 VN-3398 VN-3403 VN-3408 VN-3410 VN-3411 VN-3412 VN-3415 VN-3418 VN-3419 VN-3426 VN-3442 VN-3443 VN-3444 VN-3446 VN-3448 VN-3451 VN-3454 VN-3457 VN-3461 VN-3465 VN-3467

227 228 229 171 230 231 230 232 233 234 235 236 237 238 239 239 240 241 242 250 243 244 234 244 245 246

I I I IIA I I I I I I I I I I I I I IIA I I I I I I I I

Clonal Complex (SLV-Level) Singleton Singleton 1 Singleton Singleton Singleton Singleton Singleton 1 Singleton Singleton Singleton Singleton Singleton Singleton Singleton 1 Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton

Clonal Complex (TLV-Level) Singleton Singleton 2 Singleton Singleton Singleton Singleton Singleton 2 Singleton Singleton Singleton Singleton Singleton 4 4 2 Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton Singleton

glp gyrB mdh metG purM dtdS lysA pntA pyrC tnaA 7 71 19 55 72 19 72 24 19 19 7 8 73 9 74 74 19 35 75 26 76 10 19 10 47 18

23 12 61 1 60 42 60 23 61 42 11 62 15 23 11 11 61 63 42 2 64 28 42 28 14 19

2 2 73 4 2 13 2 70 2 13 11 2 16 11 2 2 73 3 2 2 2 13 13 13 17 17

22 41 60 5 24 12 24 12 60 62 22 2 7 24 22 22 60 25 12 3 60 7 62 7 17 17

8 8 65 30 9 8 9 9 65 40 59 13 9 8 60 60 65 25 9 62 8 8 40 8 8 8

22 22 100 66 22 22 22 35 100 82 94 19 35 96 103 103 100 105 22 110 106 107 82 107 44 5

4 69 96 42 12 104 12 12 96 12 65 75 91 56 12 12 96 114 12 12 98 26 12 26 18 95

66 67 80 10 13 21 13 68 80 13 69 9 13 11 20 20 80 36 70 20 13 71 13 71 21 21

56 56 12 37 84 7 84 80 11 64 12 5 33 12 81 81 11 43 82 84 83 15 64 15 4 4

18 73 76 53 18 49 18 30 76 15 51 18 9 9 50 50 76 77 78 83 79 80 15 80 9 26


S9 Table S3. Cont.

Strain ID

MLST ST

MLST-Cluster


247 248 244 249 250 240 252 253 254 255 255 256 110 257 258 259 133 287 260 261 262 263 113 264 265 266

I IIA I I I I I I I I I I IIA I I I I I I I IIB IIB I I I I

Clonal Complex (SLV-Level) Singleton Singleton Singleton Singleton Singleton 1 Singleton Singleton Singleton Singleton Singleton Singleton Singleton 7 Singleton Singleton 6 2 Singleton Singleton Singleton 3 4 Singleton Singleton Singleton

Clonal Complex (TLV-Level) Singleton Singleton Singleton Singleton Singleton 2 Singleton Singleton Singleton Singleton Singleton Singleton Singleton 9 4 Singleton 3 5 Singleton Singleton Singleton 6 7 Singleton 1 Singleton


23 65 28 66 2 61 67 28 16 23 23 43 1 14 26 68 14 14 2 12 39 13 8 14 41 38

2 71 13 44 2 73 2 11 75 9 9 9 12 15 2 73 15 11 20 74 14 14 10 11 10 11

12 63 7 64 3 60 22 23 2 12 12 12 31 40 22 22 7 61 2 22 42 7 7 7 3 23

61 23 8 59 62 65 9 8 9 63 63 63 27 9 60 65 9 8 8 8 25 25 9 9 9 9

22 108 107 109 110 100 22 34 111 111 111 22 23 40 103 112 34 64 107 2 24 27 22 113 114 34

113 41 26 100 12 96 12 60 17 64 64 93 36 6 12 112 6 53 110 6 92 61 33 105 33 110

7 72 71 69 20 80 73 21 18 74 74 74 1 25 75 25 15 23 71 76 41 77 9 78 15 5

26 59 15 11 84 11 26 5 85 9 9 86 48 5 81 11 14 17 87 50 13 13 5 88 49 17

62 81 80 82 83 76 10 52 45 84 84 85 47 4 18 18 14 41 86 87 11 11 30 39 44 49


S10 Table S3. Cont.

Strain ID

MLST ST

MLST-Cluster


226 268 217 251 269 268 268 270 271 226 272 144 273 274 226 275 126 133 269 128 113 276 128 132 277 278

IIB I IIB I I I I I I IIB IIB I I I IIB I I I I IIB I I IIB I I I

Clonal Complex (SLV-Level) Singleton Singleton Singleton 2 Singleton Singleton Singleton Singleton Singleton Singleton 3 Singleton 5 Singleton Singleton Singleton Singleton 6 Singleton Singleton 4 4 Singleton Singleton Singleton Singleton

Clonal Complex (TLV-Level) Singleton 3 Singleton 5 1 3 3 1 Singleton Singleton 6 Singleton 1 Singleton Singleton Singleton 1 3 1 Singleton 7 7 Singleton 8 8 Singleton


13 14 39 14 8 14 14 40 41 13 13 14 8 40 13 14 8 14 8 13 8 8 13 40 40 42

42 15 42 11 10 15 15 10 11 42 14 2 10 2 42 11 10 15 10 14 10 10 14 44 44 11

42 7 29 61 3 7 7 3 23 42 40 7 3 65 42 2 3 7 3 13 7 7 13 7 2 7

23 9 57 8 9 9 9 9 8 23 25 41 9 13 23 3 9 9 9 12 9 9 12 8 8 8

67 5 27 99 115 5 5 22 64 67 27 19 115 81 67 116 22 34 115 4 22 22 4 22 22 82

103 6 41 53 17 6 6 33 59 103 61 17 41 1 103 62 55 6 17 41 33 33 41 57 58 57

13 15 77 23 15 15 15 15 5 13 77 46 15 46 13 5 15 15 15 1 9 9 1 15 5 65

13 14 13 17 49 14 14 49 5 13 13 57 15 5 13 53 49 14 49 13 5 5 13 5 5 5

11 45 11 41 44 45 45 44 49 11 11 30 44 44 11 25 44 14 44 11 30 30 11 50 50 45


S11 Table S3. Cont.

Strain ID

MLST ST

MLST-Cluster

VN-3972 VN-3973 VN-3974 VN-3975 VN-3976 VN-3977 VN-3978 VN-3979 VN-3980 VN-3981 VN-3982 VN-5163

277 268 279 280 281 281 282 128 269 283 284 65

I I I I IIB IIB I IIB I I I I

Clonal Complex (SLV-Level) Singleton Singleton Singleton 5 Singleton Singleton Singleton Singleton Singleton Singleton 6 7

Clonal Complex (TLV-Level) Singleton 3 Singleton 1 Singleton Singleton Singleton Singleton 1 Singleton 3 9

glp gyrB mdh metG purM dtdS lysA pntA pyrC tnaA 49 13 47 8 4 4 52 12 8 45 13 13

40 14 69 8 13 13 42 13 8 2 14 14

44 15 46 10 14 14 2 14 10 20 15 15

2 7 7 66 29 29 7 13 3 2 7 7

8 9 3 9 43 43 8 12 9 8 9 9

MLST, multilocus sequence typing; ST, sequence type; SLV, single locus variant; TLV, triple locus variant.

22 5 107 115 24 24 35 4 115 60 34 40

58 6 108 41 109 109 4 41 17 26 6 6

5 15 79 15 32 32 5 1 15 20 15 25

5 14 5 15 13 13 4 13 49 17 14 5

50 45 88 44 11 11 4 11 44 43 39 4


S12

Table S4. Primers and probes used for PCR amplification and sequencing. Primer Name SerE-F SerE-R Bt2-F Bt2-R vvhA-F vvhA-R UtoxF VvtoxR vcg-typeC-F vcg-typeE-F vcg-typeC/E-R VVA1612F VVA1613R VVA1634F VVA1634R VVA1612bF VVA1637R VVA1633a_F a VVA1635c_R a VVA1633b_F a VVA1635a_R a VVA1635b_R a VVA1634a_F a VVA1634a_R a VVA1634b_F a VVA1634b_R a

Specificity/Gene Target/Designation specific for serovar E specific for serovar E specific for biotype 2 specific for biotype 2 V. vulnificus-specific hemolysin V. vulnificus-specific hemolysin toxR V. vulnificus-specific toxR virulence correlated gene clinical allele virulence correlated gene environmental allele virulence correlated gene Region XII, 5′flanking region Region XII, chondroitinase AC lyase Region XII, arylsulfatase A Region XII, arylsulfatase A Region XII, 5′flanking region Region XII, 3′flanking region Region XII Region XII Region XII Region XII Region XII Region XII, arylsulfatase A Region XII, arylsulfatase A Region XII, arylsulfatase A Region XII, arylsulfatase A

Sequence (5′ to 3′) TGTTGTTCTTGCCCACTCTC CGCGCTTAGATTTGTCTCACC AGAGATGGAAGAAACAGGCG GGACAGATATAAGGGCAAATGG CGCCACCCACTTTCGGGCC CCGCGGTACAGGTTGGCGC GASTTTGTTTGGCGYGARCAAGGTT AACGGAACTTAGACTCCGAC AGCTGCCGATAGCGATCT CTCAATTGACAATGATCT CGCTTAGGATGATCGGTG ACCCTGATCGTTGGCTACTC GGAGCGGTGTGATGGTGTTG TGACACCCAACCTAGACCAC ATTGATGCCAACCTGAG TGTGGAGAGCGGCAAGATCAAG AACATCAACCAGCGAGTCGAAC CGTCATCACTCATGTCAAAGC GGTTTCATCGTCCCAAATGG TCGAGATTGCAAACCGGACC CGGCGTAGAGAATGATAACG CGTACATCACATCCAACAGTTC GGCACGTTCGATCAACATTG TGATCGAACGTGCCATAGCC TCTATTTCGCCAACGTGAC GCAAAGTAATCGCGGATCTTG

Amplicon (bp) 665

Ta (ºC) 64

344 519 435

60

277

56 47

2.257

65

1.364

55

1.200

65

2483

60

Reference [1] [1] [1] [1] [1] [1] [2] [2] [3] [3] [3] [4] [4] [4] [4] [4] [4] this study this study this study this study this study this study this study this study this study


S13 Table S4. Cont.

Primer Name VVA1634c_F a VVA1634d_F a Vvu16S51-F b Vvu16S221-R b Vvu16SA-P b (2091859) Vvu16SB-P b (2091860) nanA-F nanA -R Man IIA F Man IIA R

Specificity/Gene Target/Designation Region XII, arylsulfatase A Region XII, arylsulfatase A 16S rRNA gene 16S rRNA gene 16S rRNA gene type A allele 16S rRNA gene type B allele sialic acid catabolism (SAC) cluster, aldolase, sialic acid catabolism (SAC) cluster, aldolase, mannitol fermentation operon, enzyme IIA mannitol fermentation operon, enzyme IIA

Sequence (5′ to 3′) CCCCTATCAAACCAACAACC GCTGCTTTACCGATGTGCTC CAAGTCGAGCGGCAGCA TCCTGACGCGAGAGGCC 6-FAM-TGATAGCTTCGGCTCAAMGBNFQ VIC-CCCGTAGGCATCATGCMGBNFQ TKATCGCCGCTCCYCATACA GCAACGCCACCGTATTCAAC GATGTTGGTGAACAACTTCTCTGC TCTGAAGCCTGTTGGATGCC

Amplicon (bp)

Ta (ºC)

171

62

Ta, annealing temperature. a used for gene sequencing; b used for Real-Time PCR.

Reference this study this study [5] [5]

probe

[5]

probe

[5]

745

58

243

61

[6] [7] [8] [8]


S14

Table S5. Genotypic and phenotypic characteristics of V. vulnificus strains from the Baltic Sea and North Sea. Strain ID

Source

Sampling Site No. a

BT b

Serum Resistance

VN-0279 VN-2813 VN-2814 VN-2961 VN-2969 VN-3363 VN-3364 VN-3366 VN-3367 VN-3368 VN-3369 VN-3373 VN-3374 VN-3378 VN-3379 VN-3394 VN-3398 VN-3403 VN-3408 VN-3410 VN-3411 VN-3412 VN-3415

B-sw N-sw N-sw B-sw B-sw N-sd N-sd N-sd N-sw N-sw N-sw N-sd N-sd N-sw N-sw N-sd N-sw N-sw N-sw N-sw N-sd N-sw N-sw

12 − − 11 10 4 4 4 7 7 7 5 5 2 2 8 8 8 2 4 4 7 7

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

R R R R R R R R R I R R R R R R R R R R I R R

Mannitol Fermentation c − + + + + + + − + + + − + − + + + + + − + + −

16S rRNA type

vcg Type

Region XII

nanA

MLST -ST

MLST Cluster

Risk Group d

AB A A B AB A A A A B B A A A B A A A A A A A A

E E E E E E E E E C C E E E C E E E E E E E E

+ − − + + + + − − − − − − − + + + + + − − − −

+ + + − − − − − + + + − + + + + + + + + + + +

217 219 219 220 220 223 223 224 219 225 225 227 228 229 171 230 231 230 232 233 234 235 236

IIB I I IIB IIB I I I I IIA IIA I I I IIA I I I I I I I I

2 2 2 2 2 2 2 1 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2


S15 Table S5. Cont.

Strain ID

Source

Sampling Site No. a

BT b

Serum Resistance


N-sd N-sw N-sd N-sd N-sd N-sd N-sd N-sw N-sw N-sd N-sd N-sw N-sd N-sd N-sw N-sd N-sd N-sw N-sw N-sd N-sd N-sw N-sw

7 5 5 1 4 4 7 7 7 7 7 5 5 5 7 7 5 7 7 4 7 5 9

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

R R R R R R I R I S R S R R I R S R R R R R R

Mannitol Fermentation c + + + + − + + + − − + − − − + + − + + − + − −

16S rRNA Type

vcg Type

Region XII

nanA

MLSTST

MLST Cluster

Risk Group d

A A A A A B A A A A A A A A A B A A A A A A A

E E E E E C E E E E E E E E C C E E E E E E E

+ − + + − + − + − − − − − − + − − − + − + − −

+ + + + + − + + − − + − − − + + − − + + + + −

237 238 239 239 240 241 242 250 243 244 234 244 245 246 247 248 244 249 250 240 252 253 254

I I I I I IIA I I I I I I I I I IIA I I I I I I I

2 2 2 2 2 2 2 2 1 1 2 1 1 1 2 2 1 2 2 2 2 2 1


S16 Table S5. Cont.

Strain ID

Source

Sampling Site No. a

BT b

Serum Resistance


N-sw N-sw N-sw N-sw N-sw N-sw N-sw B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sw B-sd B-sd B-sd B-sd B-sw

6 8 9 6 3 8 9 20 20 19 18 18 17 16 16 26 22 21 21 13 26 22 21

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

R R R R S R R R I R R R R I R I I R R R R I R

Mannitol Fermentation c + + + + − + − − − − − − − − − − − − − − − − −

16S rRNA Type

vcg Type

Region XII

nanA

MLST -ST

MLST Cluster

Risk Group d

A A A B A A A A A A A AB AB A A A A AB A AB A A A

E E E E E E E E E E E E E E E E E E E E E E E

+ − + + − + − − − − − + + − − − − + − + − − −

+ + + + − + − − − − − + − − − − − + − + − − −

255 255 256 110 257 258 259 133 287 260 261 262 263 113 264 265 266 226 268 217 251 269 268

I I I IIA I I I I I I I IIB IIB I I I I IIB I IIB I I I

2 2 2 2 1 2 1 1 1 1 1 2 2 1 1 1 1 2 1 2 1 1 1


S17 Table S5. Cont.

Strain ID

Source

Sampling Site No. a

BT b

Serum Resistance


B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sd B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw B-sw

21 21 20 18 17 17 22 22 19 23 24 24 23 27 27 27 27 27 23 25 24 14 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

R R R R R R I R R R S R S R I I R R R R R R I

Mannitol Fermentation c − − − − − − − − − − − − − − − − − + + + + − −

16S rRNA Type

vcg Type

Region XII

nanA

MLSTST

MLST Cluster

Risk Group d

A A A AB AB A A A AB A A A A AB A A AB A A A A A A

E E E E E E E E E E E E E E E E E E E E E E E

− − − + + − − − + − − − − + − − + + + − + − −

− − − + − − − − + − − − − + − − + + − − − − −

268 270 271 226 272 144 273 274 226 275 126 133 269 128 113 276 128 132 277 278 277 268 279

I I I IIB IIB I I I IIB I I I I IIB I I IIB I I I I I I

1 1 1 2 2 1 1 1 2 1 1 1 1 2 1 1 2 2 2 2 2 1 1


S18 Table S5. Cont.

Strain ID

Source

Sampling Site No. a

BT b

Serum Resistance

VN-3975 VN-3976 VN-3977 VN-3978 VN-3979 VN-3980 VN-3981 VN-3982 VN-5163

B-sw B-sw B-sw B-sw B-sd B-sw B-sw B-sw B-sw

15 15 15 15 15 27 23 27 28

1 1 1 1 1 1 1 1 1

R R R R R R R R S

Mannitol Fermentation c − − − + − − − − −

16S rRNA Type

vcg Type

Region XII

nanA

MLST -ST

MLST Cluster

Risk Group d

A AB AB A AB A A A A

E E E E E E E E E

− + + − + − − − −

− + + − + − − − −

280 281 281 282 128 269 283 284 65

I IIB IIB I IIB I I I I

1 2 2 2 2 1 1 1 1

N, North Sea; B, Baltic Sea; sw, seawater; sd, sediment; R, resistant; I, intermediate resistant; S, susceptible; ST, sequence type. a Sampling site numbers shown in Figure 1. b Biotype assessed biochemically and by multiplex PCR. c Mannitol fermentation tested biochemically and by presence of mannitol fermentation operon (PCR). d Risk group 2 comprising strains with two or more pathogenicity markers, risk group 1 comprising strains without or with one pathogenicity marker.


S19

Figure S1. Population structure of V. vulnificus biotype 1 isolates from the North Sea (▲) and Baltic Sea (●) based on concatenated MLST sequences of three housekeeping genes (gyrB, dtdS, and pyrC). Bootstrap values above 70% are shown next to the branches. Semicircles around the tree highlight the association of strains to MLST cluster I (white), IIA (grey), and IIB (black). Sequences from clinical (□) and environmental (○) Baltic Sea isolates from a previous study [6] were included for comparison. References 1 2 3

Sanjuan, E.; Amaro, C. Multiplex PCR assay for detection of vibrio vulnificus biotype 2 and simultaneous discrimination of serovar e strains. Appl. Environ. Microbiol. 2007, 73, 2029–2032. Bauer, A.; Roervik, L.M. A novel multiplex pcr for the identification of vibrio parahaemolyticus, vibrio cholerae and vibrio vulnificus. Lett. Appl. Microbiol. 2007, 45, 371–375. Rosche, T.M.; Yano, Y.; Oliver, J.D. A rapid and simple PCR analysis indicates there are two subgroups of vibrio vulnificus which correlate with clinical or environmental isolation. Microbiol. Immunol. 2005, 49, 381–389.

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5

6

7 8

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Cohen, A.L.V.; Oliver, J.D.; DePaola, A.; Feil, E.J.; Boyd, E.F. Emergence of a virulent clade of vibrio vulnificus and correlation with the presence of a 33-kilobase genomic island. Appl. Environ. Microbiol. 2007, 73, 5553–5565. Vickery, M.C.L.; Nilsson, W.B.; Strom, M.S.; Nordstrom, J.L.; DePaola, A. A real-time pcr assay for the rapid determination of 16s rrna genotype in vibrio vulnificus. J. Microbiol. Methods 2007, 68, 376–384. Bier, N.; Bechlars, S.; Diescher, S.; Klein, F.; Hauk, G.; Duty, O.; Strauch, E.; Dieckmann, R. Genotypic diversity and virulence characteristics of clinical and environmental vibrio vulnificus isolates from the baltic sea region. Appl. Environ. Microbiol. 2013, 79, 3570–3581. Lubin, J.B.; Kingston, J.J.; Chowdhury, N.; Boyd, E.F. Sialic acid catabolism and transport gene clusters are lineage specific in vibrio vulnificus. Appl. Environ. Microbiol. 2012, 78, 3407–3415. Froelich, B.; Oliver, J. Orientation of mannitol related genes can further differentiate strains of vibrio vulnificus possessing the vcgc allele. Adv. Stud. Biol. 2011, 3, 151–160.

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