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

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 (stx_1c+stx_2d; ONT:H8); 2, 3115/97 (stx_1c+stx_2d; O128:H2); 3, 521/99 (stx_1c+stx_2d; O^rough:H19); 4, 4756/98 (stx_1c+stx_2d; O70:H⁻); 5, 273/00 (stx_1c+stx_2d; O128:H⁻; sheep); 6, 295/00 (stx_1c+stx_2d; 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+stx_2c; O157:H⁻); 12, 2050/98 (stx₁+stx₂+stx_2c; O157:H⁻).

TABLE 1.

PCR primers and conditions used in this study

Primer pair	Sequence	Target	PCR conditions^a			Length of PCR product (bp)	Reference
Primer pair	Sequence	Target	Denaturing	Annealing	Extension	Length of PCR product (bp)	Reference
KS7	5′-CCC GGA TCC ATG AAA AAA ACA TTA TTA ATA GC-3′	stxB ₁	94°C, 30 s	52°C, 60 s	72°C, 40 s	282	26
KS8	5′-CCC GAA TTC AGC TAT TCT GAG TCA ACG-3′	stxB _1c
GK3	5′-ATG AAG AAG ATG TTT ATG-3′	stxB ₂	94°C, 30 s	52°C, 60 s	72°C, 40 s	260	13
GK4	5′-TCA GTC ATT ATT AAA CTG-3′	stxB _2c
VT2-cm	5′-AAG AAG ATA TTT GTA GCG G-3′	stxB _2d	94°C, 30 s	55°C, 60 s	72°C, 60 s	256	24
VT2-f	5′-TAA ACT GCA CTT CAG CAA AT-3′
SK1	5′-CCC GAA TTC GGC ACA AGC ATA AGC-3′	eae	94°C, 30 s	52°C, 60 s	72°C, 60 s	863	26
SK2	5′-CCC GGA TCC GTC TCG CCA GTA TTC G-3′
Stx1c-1^b	5′-TTT TCA CAT GTT ACC TTT CCT-3′	stxA _1c	94°C, 30 s	51°C, 60 s	72°C, 60 s	498	This study
Stx1c-2	5′-CAT AGA AGG AAA CTC ATT AGG-3′
Oligo 1	5′-TCG CAT GAG ATC TGA CC-3′	stx ₁, stx_1c	94°C, 30 s^c	55°C, 60 s	72°C, 90 s	1,469	22
Oligo 2	5′-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 stx_1c 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 strains^a investigated in this study

stx genotype	No. of isolates	Serotype (no. of isolates)						Vero cell cyto- toxicity titer		VTEC-RPLA titer with:
		Serotype (no. of isolates)						Vero cell cyto- toxicity titer		Stx1 reagent		Stx2 reagent
		O157:H7/H⁻	O26:H11/H⁻	O103:H2/H⁻	O111:H8/H⁻	O145:H⁻	Others	Range	Median	Range	Median	Range	Median
stx ₁ ^b	105	1	21	25	10	11	37^c	16-512	64	16-256	64	<2	<2
stx _1c ^b	12	0	0	0	0	0	12^d	32-256	64	2-8	4	<2	<2
stx ₁+stx₂	49	25	12	1	7	1	3^e	32-2,048	256	16-256	64	32-256	128
stx _1c+stx₂	1	0	0	0	0	0	1^f	256	256	8	8	128	128
stx ₁+stx_2c	14	10	0	0	0	1	3^g	16-128	48	8-128	32	4-32	16
stx ₁+stx₂+stx_2c	4	2	0	0	0	0	2^h	64-256	128	8-64	24	32-128	64
stx ₁+stx_2d	4	0	0	0	0	0	4ⁱ	16-128	64	16-32	24	4	4
stx _1c+stx_2d	25^j	0	0	0	0	0	25^k	16-128	64	2-8	4	4-8	6

↵ a All but two STEC isolates were of human origin; two strains were isolated from sheep.
↵ b Both stx₁ and stx_1c 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), O^rough:H⁻ (five strains), O^rough:HNT.
↵ d O8:H19, O8:H⁻, O74:H⁻, O75:H33, O78:H⁻ (three strains), O112:H⁻, O128:H⁻, ONT:H⁻ (two strains), O^rough:HNT.
↵ e O4:H⁻, O68:H4, O118:H⁻.
↵ f ONT:H⁻.
↵ g O104:H16, O113:H⁻, O120:H⁻.
↵ h O75:H⁻, O^rough:H25.
↵ i O62:H⁻ (two strains), O91:H⁻, O128:H⁻.
↵ j Twenty-three of 25 STEC strains of the stx_1c+stx_2d 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), O^rough:H19, O^rough:HNT (three strains).