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Bacteriology

Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans

Wenlan Zhang, Martina Bielaszewska, Thorsten Kuczius, Helge Karch
Wenlan Zhang
1Institut für Hygiene, Universitätsklinikum Münster, 48149 Münster
2Institut für Hygiene und Mikrobiologie der Universität Würzburg, 97080 Würzburg, Germany
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Martina Bielaszewska
1Institut für Hygiene, Universitätsklinikum Münster, 48149 Münster
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Thorsten Kuczius
1Institut für Hygiene, Universitätsklinikum Münster, 48149 Münster
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Helge Karch
1Institut für Hygiene, Universitätsklinikum Münster, 48149 Münster
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  • For correspondence: hkarch@uni-muenster.de
DOI: 10.1128/JCM.40.4.1441-1446.2002
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    FIG. 1.

    Agarose gel electrophoresis of KS7-KS8 PCR products digested with FspI (A) or HhaI (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 (stx1c+stx2d; ONT:H8); 2, 3115/97 (stx1c+stx2d; O128:H2); 3, 521/99 (stx1c+stx2d; Orough:H19); 4, 4756/98 (stx1c+stx2d; O70:H−); 5, 273/00 (stx1c+stx2d; O128:H−; sheep); 6, 295/00 (stx1c+stx2d; O128:H−; sheep); 7, EDL 933 (stx1+stx2; O157:H7); 8, 2544/00 (stx1; O145:H−); 9, 3385/00 (stx1+stx2; O111:H−); 10, 4424/99 (stx1+stx2; O157:H7); 11, 2049/98 (stx1+stx2c; O157:H−); 12, 2050/98 (stx1+stx2+stx2c; O157:H−).

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  • TABLE 1.

    PCR primers and conditions used in this study

    Primer pairSequenceTargetPCR conditionsaLength of PCR product (bp)Reference
    DenaturingAnnealingExtension
    KS75′-CCC GGA TCC ATG AAA AAA ACA TTA TTA ATA GC-3′ stxB 1 94°C, 30 s52°C, 60 s72°C, 40 s282 26
    KS85′-CCC GAA TTC AGC TAT TCT GAG TCA ACG-3′ stxB 1c
    GK35′-ATG AAG AAG ATG TTT ATG-3′ stxB 2 94°C, 30 s52°C, 60 s72°C, 40 s260 13
    GK45′-TCA GTC ATT ATT AAA CTG-3′ stxB 2c
    VT2-cm5′-AAG AAG ATA TTT GTA GCG G-3′ stxB 2d 94°C, 30 s55°C, 60 s72°C, 60 s256 24
    VT2-f5′-TAA ACT GCA CTT CAG CAA AT-3′
    SK15′-CCC GAA TTC GGC ACA AGC ATA AGC-3′ eae 94°C, 30 s52°C, 60 s72°C, 60 s863 26
    SK25′-CCC GGA TCC GTC TCG CCA GTA TTC G-3′
    Stx1c-1b5′-TTT TCA CAT GTT ACC TTT CCT-3′ stxA 1c 94°C, 30 s51°C, 60 s72°C, 60 s498This study
    Stx1c-25′-CAT AGA AGG AAA CTC ATT AGG-3′
    Oligo 15′-TCG CAT GAG ATC TGA CC-3′ stx 1, stx1c94°C, 30 sc55°C, 60 s72°C, 90 s1,469 22
    Oligo 25′-AAC TGA CTG AAT TGA GAT G-3′Whole
    • ↵ a All PCRs included 30 cycles followed by a final extension step of 5 min at 72°C.

    • ↵ b Primers Stx1c-1 and Stx1c-2 were derived from the positions 511 to 531 and 988 to 1008, respectively, of the stx1c sequence from strain 3115/97 determined in this study (accession no. AJ312232 ).

    • ↵ c PCR conditions were established in this study.

  • TABLE 2.

    stx genotypes, serotypes, and Stx production by 214 STEC strainsa investigated in this study

    stx genotypeNo. of isolatesSerotype (no. of isolates)Vero cell cyto- toxicity titerVTEC-RPLA titer with:
    Stx1 reagentStx2 reagent
    O157:H7/H−O26:H11/H−O103:H2/H−O111:H8/H−O145:H−OthersRangeMedianRangeMedianRangeMedian
    stx 1 b 10512125101137c16-5126416-25664<2<2
    stx 1c b 120000012d32-256642-84<2<2
    stx 1+stx24925121713e32-2,04825616-2566432-256128
    stx 1c+stx21000001f25625688128128
    stx 1+stx2c141000013g16-128488-128324-3216
    stx 1+stx2+stx2c4200002h64-2561288-642432-12864
    stx 1+stx2d4000004i16-1286416-322444
    stx 1c+stx2d 25j0000025k16-128642-844-86
    • ↵ a All but two STEC isolates were of human origin; two strains were isolated from sheep.

    • ↵ b Both stx1 and stx1c genes were originally detected as stx1 by PCR with primer pair KS7-KS8, without restriction.

    • ↵ c O3:H2, O3:H10, O8:H− (nonmotile) (three strains), O25:H− (two strains), O31:H−, O62:H−, O84:H4, O84:H− (three strains), O91:H14 (two strains), O92:H33, O112:H−, O118:H− (two strains), O119:H2, O128:H−, O129:H−, O146:H20, O152:H4, O156:H−, ONT (nontypeable):H14 (two strains), ONT:H− (four strains), Orough:H− (five strains), Orough:HNT.

    • ↵ d O8:H19, O8:H−, O74:H−, O75:H33, O78:H− (three strains), O112:H−, O128:H−, ONT:H− (two strains), Orough:HNT.

    • ↵ e O4:H−, O68:H4, O118:H−.

    • ↵ f ONT:H−.

    • ↵ g O104:H16, O113:H−, O120:H−.

    • ↵ h O75:H−, Orough:H25.

    • ↵ i O62:H− (two strains), O91:H−, O128:H−.

    • ↵ j Twenty-three of 25 STEC strains of the stx1c+stx2d genotype were isolated from humans; two strains (both of serotype O128:H−) originated from sheep.

    • ↵ k O22:H8, O70:H−, O75:H8, O75:H21, O96:H−, O113:H− (two strains), O128:H2 (five strains), O128:H− (three strains), O128:HNT, ONT:H8 (three strains), ONT:H− (two strains), Orough:H19, Orough:HNT (three strains).

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Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans
Wenlan Zhang, Martina Bielaszewska, Thorsten Kuczius, Helge Karch
Journal of Clinical Microbiology Apr 2002, 40 (4) 1441-1446; DOI: 10.1128/JCM.40.4.1441-1446.2002

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Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans
Wenlan Zhang, Martina Bielaszewska, Thorsten Kuczius, Helge Karch
Journal of Clinical Microbiology Apr 2002, 40 (4) 1441-1446; DOI: 10.1128/JCM.40.4.1441-1446.2002
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KEYWORDS

Escherichia coli
Genetic Variation
Shiga Toxin 1

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