3. Materials and Methods
3.3. Microbiological methods
3.3.1. Bacterial strains and plasmids
In Table 8 and Table 9 bacterial strains and plasmids used in this study are listed. The additional Y. enterocolitica strains tested for validation of the patho-serotyping PCR are listed in Table 10.
Table 8 Bacterial strains used in this study.
(FBI-Zoo: Food-Borne Zoonotic Infections of Humans, BMBF project; FLI: Friedrich-Löffler Institute; MvPI:
Max-von-Pettenkofer Institute) Y. enterocolitica strains
Bacterial strain ID Features Reference
8081 Bioserotype 1B/O:8, American human isolate (Portnoy et al., 1981)
WA-314 (ATCC 51871) Bioserotype 1B/O:8, clinical isolate (Heesemann and Laufs, 1983) Y11 (DSMZ 13030) Bioserotype 4/O:3, human isolate (Neubauer et al., 2000a) W22703 Bioserotype 2/O:9, mutant from strain W227 (Cornelis and Colson, 1975)
Y5.27P Bioserotype 3/O:5,27 MvPI collection
NF-O Bioserotype 1A/O:5, clinical isolate (Ratnam et al., 1982) IP2222 Bioserotype 1A/O:36, Japanese isolate from
water (Grant et al., 1999)
Y8265/556 Bioserotype 4/O:3, French human isolate MvPI collection Y5307/647 Bioserotype 4/O:3, Finnish human isolate MvPI collection
P1 (BR6319) Bioserotype O:3, German isolate from pig Prof. Neubauer, FLI Jena P4 (BR0055) Bioserotype O:3,German isolate from pig Prof. Neubauer, FLI Jena 149 (CN-Y641) Bioserotype 4/O:3, Belgian isolate from puppy MvPI collection
150 (CN-Y486) Bioserotype 4/O:3, Belgian isolate form calf MvPI collection D1 (M1-S3405) Bioserotype 4/O:3, German human isolate MvPI collection D2 (M2-S3547) Bioserotype 4/O:3,German human isolate,
obtained 45 days after D1 from same patient MvPI collection D3 (CN-35) Bioserotype 4/O:3, German human isolate MvPI collection D4 (CN-142) Bioserotype 4/O:3, Belgian isolate from pig MvPI collection D5 (CN-146) Bioserotype 4/O:3, Danish isolate from pig MvPI collection D6 (CN-243/Y745) Bioserotype 3/O:3, Japanese isolate MvPI collection D7 (P8/FBI-00033) Bioserotype 4/O:3, German isolate from pig FBI-Zoo collection D8 (P14/FBI-03871) Bioserotype 4/O:3, German isolate from pig FBI-Zoo collection D9 (P15/04247) Bioserotype 4/O:3, German isolate from pig FBI-Zoo collection
Other strains
Species Features Reference
E. coli T7 express (BL21 derivative, no λ-prophage)
fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA1
D(mcrCmrr)114::IS10
New England Biolabs, Frankfurt
Y. frederiksenii CN-113 Strain H56-36/81, serotype O:60 MvPI collection Y. intermedia CN-114 Strain H9-36/83, serotype O:17 MvPI collection Y. kristensenii CN-115 Strain H17-36/83, serotype O:12.25 MvPI collection Y. rohdei CN-116 Strain H274-36/85, serotype O:76 MvPI collection Y. bercovieri CN-117 Strain H632-36/85, serotype O:16 MvPI collection Y. mollaretii CN-118 Strain H279-36/86, serotype O:59 MvPI collection Y. pseudotuberculosis Strain IP32953, serotype O:1 (Chain et al., 2004)
Y. pestis CN-691 Strain KIM, biotype Medievalis (Fetherston and Perry, 1994)
E. coli Strain UTI89, serotype O18:K1:H7 Prof. Stecher, MvPI
E. coli Strain CFT073, serotype O6:H1 Prof. Stecher, MvPI
S. enterica subsp.I Strain C5, serovar Typhimurium Prof. Stecher, MvPI S. enterica subsp.I Strain ATCC 14028, serovar Typhimurium Prof. Stecher, MvPI
C. jejuni Strain L11, serotype Lior 11 MvPI collection
C. jejuni Strain G23 MvPI collection
L. pneumophila Strain 2020 Prof. Hilbi, MvPI
Table 9 List of plasmids.
Plasmid Features Reference
pET-30a Expression vector, T7 promoter, N-ter and C-ter
His·Tag, KanR Novagen, Darmstadt
pET-30b Expression vector, T7 promoter, N-ter and C-ter
His·Tag, KanR Novagen, Darmstadt
pET-30c Expression vector, T7 promoter, N-ter and C-ter
His·Tag, KanR Novagen, Darmstadt
Table 10 Y. enterocolitica strains used for validation of the patho-serotyping PCR-based method.
When necessary, re-serotyping of these strains was performed by Dr. Bader (MvPI, Munich).
(CVUA: Chemisches und Veterinäruntersuchungsamt Stuttgart; FBI-Zoo: Food-Borne Zoonotic Infections of Humans, BMBF project; FLI: Friedrich-Löffler-Institute; MvPI: Max-von-Pettenkofer Institute; RKI:
Robert Koch Institute, Wernigerode)
Strain ID Features Source
CN-32 Serotype 0:3, strain 108P MvPI collection
CN-36 Bioserotype 1A/O:5, human isolate MvPI collection CN-38 Bioserotype 1A/O:4,33, isolate from milk MvPI collection CN-39 Bioserotype 1A/O:7,8, isolate from milk MvPI collection CN-40 Bioserotype 1A/O:6.30, isolate from milk MvPI collection CN-41 Bioserotype 1A/O:10,K1, isolate from milk MvPI collection
CN-42 Bioserotype 2/O:5,27 MvPI collection
CN-44 Bioserotype 4/O:9, human isolate MvPI collection CN-45 Bioserotype 4/O:5,27, human isolate MvPI collection CN-47 Bioserotype 3/O:5,27, human isolate MvPI collection CN-49 Bioserotype 2/O:5,27, human isolate MvPI collection
CN-52 Bioserotype 1A/O:48 MvPI collection
CN-53 Bioserotype 1A/O:41,43 MvPI collection
CN-54 Bioserotype 1A/O:5 MvPI collection
CN-55 Bioserotype 1B/O:8, strain CDC 544 MvPI collection CN-56 Bioserotype 1B/O:8, strain CDC 540/84 MvPI collection CN-57 Bioserotype 1B/O:8, strain E 661 Montana MvPI collection CN-58 Bioserotype 1B/O:13, strain USA 329/82 MvPI collection CN-59 Bioserotype 1B/O:20, strain CDC 3039/85 MvPI collection CN-60 Bioserotype 1B/O:21, strain CDC 209/84 MvPI collection
CN-101 Bioserotype 1A/O:36 MvPI collection
CN-170 Serotype O:3, strain 59/03 MvPI collection CN-233 Bioserotype 1B/O:8, strain IP636 MvPI collection CN-235 Bioserotype 2/O:9, strain IP22393 MvPI collection CN-237 Bioserotype 2-3/O:5,27, strain IP199 MvPI collection CN-238 Bioserotype 2-3/O:5,27, strain IP885 MvPI collection CN-239 Bioserotype 2-3/O:5,27, strain IP1607 MvPI collection CN-240 Bioserotype 2-3/O:5,27, strain IP22460 MvPI collection CN-242 Bioserotype 4/O:3, strain IP24231 MvPI collection CN-244 Bioserotype 4/O:3, strain IP24231 MvPI collection CN-245 Bioserotype 4/O:3, strain IP21981 MvPI collection
CN-249 Bioserotype 4/O:3, strain IP23357 MvPI collection CN-252 Bioserotype 4/O:3, strain IP7032 MvPI collection CN-254 Bioserotype 4/O:3, strain IP3692 MvPI collection CN-256 Bioserotype 4/O:3, strain IP4115 MvPI collection CN-262 Bioserotype 4/O:3, strain IP22274 MvPI collection CN-266 Bioserotype 4/O:3, strain IP23227 MvPI collection CN-272 Bioserotype 1A/O:5, strain IP124 MvPI collection CN-584 Bioserotype 1A/O:5, strain NF-O MvPI collection
260 Not typeable, strain FBI-4465 Klinikum der Universität München 267 Serotype O:3, strain FBI-4848 Klinikum der Universität München 282 Not typeable, strain FBI-5620 Klinikum der Universität München 292 Serotype O:27, strain FBI-6584 Klinikum der Universität München 343 Serotype O:9, strain FBI-5634 Klinikum der Universität München 377 Serotype O:3, strain FBI-6821 Klinikum der Universität München
H-82 Strain Ty 4726/85 MvPI collection
H-89 Strain Ty 448/87 MvPI collection
H-93 Strain Ty 822/88 MvPI collection
H-395 Serotype O:5,27 MvPI collection
H-522 Serotype O:1,2a,3, strain H 523/88 MvPI collection H-523 Serotype O:2a,2b,3, strain H 518/88 MvPI collection H-534 Bioserotype 2/O:5,27, strain D113 MvPI collection H-556 Bioserotype 4/O:3, strain 8265 MvPI collection H-568 Bioserotype 2/O:5,27, strain YE873 MvPI collection H-647 Bioserotype 4/O:3, strain 5307 MvPI collection H-678 Bioserotype 2/O:5,27, strain SZ 3801/03 MvPI collection H-679 Bioserotype 2/O:5,27, strain SZ 81/04 MvPI collection
H-744 Strain S11136 MvPI collection
H-745 Strain S11149 MvPI collection
H-746 Serotype O:3, strain S11925 MvPI collection H-747 Serotype O:3, strain S12723 MvPI collection H-748 Serotype O:3, strain S12891 MvPI collection H-749 Serotype O:3, strain S12982 MvPI collection H-750 Serotype O:3, strain S13559 MvPI collection
P2 Bioserotype 4/O:3, strain 0350, from pig Dr. Rau, CVUA, Stuttgart P3 Bioserotype 4/O:3, strain 5819, from pig Dr. Rau, CVUA, Stuttgart P5 Serotype O:3, strain BR0056, from pig Prof. Neubauer, FLI, Jena P6 Serotype O:3, strain BR0057, from pig Prof. Neubauer, FLI, Jena
P7 Serotype O:3, strain BR0058, from pig Prof. Neubauer, FLI, Jena P9 Bioserotype 4/O:3, strain FBI-03524, from pig FBI-Zoo collection P10 Bioserotype 4/O:3, strain FBI-03540, from pig FBI-Zoo collection P11 Bioserotype 4/O:3, strain FBI-03585, from pig FBI-Zoo collection P12 Bioserotype 4/O:3, strain FBI-03855, from pig FBI-Zoo collection P13 Bioserotype 4/O:3, strain FBI-03870, from pig FBI-Zoo collection P16 Bioserotype 4/O:3, strain FBI-05198, from pig FBI-Zoo collection P17 Bioserotype 4/O:3, strain FBI-05680, from pig FBI-Zoo collection
06-03972 Bioserotype 3/rough Dr. Tietze, RKI
06-04957 Bioserotype 3/rough Dr. Tietze, RKI
07-05684 Bioserotype 3/rough Dr. Tietze, RKI
08-00920 Bioserotype 1A/rough Dr. Tietze, RKI
08-02627 Bioserotype 1A/O:5 Dr. Tietze, RKI
09-05064 Bioserotype 4/O:3 Dr. Tietze, RKI
09-05074 Bioserotype 4/O:3 Dr. Tietze, RKI
09-05075 Bioserotype 4/O:3 Dr. Tietze, RKI
09-05076 Bioserotype 4/O:3 Dr. Tietze, RKI
09-05259 Bioserotype 3/rough Dr. Tietze, RKI
09-08364 Bioserotype 3/rough Dr. Tietze, RKI
10-00826 Bioserotype 1B/O:8 Dr. Tietze, RKI
10-00827 Bioserotype 1A/O:8 Dr. Tietze, RKI
11-01204 Bioserotype 3/O:5,27 Dr. Tietze, RKI
11-06758 Bioserotype 3/O:5,27 Dr. Tietze, RKI
11-07058 Bioserotype 1A/rough Dr. Tietze, RKI
11-08263 Bioserotype 3/O:5,27 Dr. Tietze, RKI
11-08388 Bioserotype 3/O:5,27 Dr. Tietze, RKI
11-08389 Bioserotype 3/O:5,27 Dr. Tietze, RKI
12-00565 Bioserotype 3/O:9 Dr. Tietze, RKI
12-00586 Bioserotype 3/O:9 Dr. Tietze, RKI
12-00587 Bioserotype 3/O:9 Dr. Tietze, RKI
12-00821 Bioserotype 1A/rough Dr. Tietze, RKI
12-01019 Bioserotype 3/O:9 Dr. Tietze, RKI
12-01336 Bioserotype 1A/O:8 Dr. Tietze, RKI
12-01511 Bioserotype 1A/O:5 Dr. Tietze, RKI
12-01512 Bioserotype 1A/O:8 Dr. Tietze, RKI
12-01527 Bioserotype 3/O:9 Dr. Tietze, RKI
12-01557 Bioserotype 1A/O:8 (re-serotyped as rough) Dr. Tietze, RKI
12-01616 Bioserotype 1A/O:8 Dr. Tietze, RKI
12-01779 Bioserotype 1A/O:5 Dr. Tietze, RKI
12-01805 Bioserotype 3/O:9 Dr. Tietze, RKI
12-01910 Bioserotype 3/O:9 Dr. Tietze, RKI
12-02272 Bioserotype 1A/O:5 Dr. Tietze, RKI
12-02439 Bioserotype 1A/O:5 Dr. Tietze, RKI
12-02966 Bioserotype 1A/O:8 Dr. Tietze, RKI
12-03062 Bioserotype 1A/O:5 Dr. Tietze, RKI
12-03266 Bioserotype 1A/rough Dr. Tietze, RKI
12-03276 Bioserotype 4/O:3 Dr. Tietze, RKI
12-03288 Bioserotype 1A/rough Dr. Tietze, RKI
12-03324 Bioserotype 1A/rough Dr. Tietze, RKI
12-03401 Bioserotype 1A/rough Dr. Tietze, RKI
12-03475 Bioserotype 4/O:3 Dr. Tietze, RKI
12-03489 Bioserotype 4/O:3 Dr. Tietze, RKI
12-03561 Bioserotype 4/O:3 Dr. Tietze, RKI
12-03562 Bioserotype 4/O:3 Dr. Tietze, RKI
12-03803 Bioserotype 3/rough Dr. Tietze, RKI
12-04470 Bioserotype 1A/rough Dr. Tietze, RKI
12-05220 Bioserotype 1B/O:8 Dr. Tietze, RKI
13-00330 Bioserotype 4/O:5,27 Dr. Tietze, RKI