Shiga Toxin, Cytolethal Distending Toxin, and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

DOI: 10.1128/JCM.00201-09

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FIG. 1.
HhaI fliC RFLP patterns of STEC O91 isolates of different serotypes and of Hnt and NM strains compared with E. coli H type strains expressing H8, H10, H14, and H21. Lane 1, E. coli H8 type strain H 515b (O103:H8), which displays the H8b fliC genotype (3); lane 2, STEC O91:H8 (06-03971); lane 3, E. coli H10 type strain E 77a (O35:H10); lane 4, STEC O91:H10 (05-06323); lane 5, E. coli H14 type strain F 10018-41 (O18ab:H14); lane 6, STEC O91:H14 (00-04243); lane 7, STEC O91:Hnt (01-04459); lane 8, STEC O91:NM (02-03777); lane 9, E. coli H21 type strain U 11a-44 (O8:H21); lane 10, STEC O91:H21 (00-04445-2); lane M, 100-bp ladder (Invitrogen).
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FIG. 2.
Photomicrographs of HBMECs after 3 days (A) and 5 days (B) of incubation with supernatant of a CDT-V-producing STEC O91:H21 strain. (C) Control HBMECs incubated 5 days in cell culture medium. Bars, 200 μm.

TABLE 1.

stx genotypes of STEC O91 isolates belonging to different serotypes

stx genotype^a	No. of strains of STEC serotype:
stx genotype^a	O91:H8 (n = 1)	O91:H10 (n = 2)	O91:H14/Hnt/NM^b (n = 77)	O91:H21 (n = 20)^c
stx ₁	0	1	69	0
stx ₁ stx _2d-EH250	0	0	6	0
stx _2d-EH250	0	0	2	0
stx ₂	0	0	0	1
stx ₂ stx _2c	0	1	0	0
stx _2dact	0	0	0	6
stx ₁ stx _2dact	0	0	0	1
stx ₂ stx _2dact	0	0	0	6
stx ₁ stx ₂ stx _2dact	0	0	0	4
stx ₁ stx _2v-O91:H21	0	0	0	1
stx ₁ stx _2v-O91:H8	1	0	0	0

↵ a stx genotyping was performed as described previously (7, 16, 17, 50), and the stx₂ variants (stx_2v-O91:H21 and stx_2v-O91:H8) were identified by sequence analysis of nontypeable stx genes. stx_2dact encodes Stx2d activatable by intestinal mucus and elastase (7, 24, 27); stx_2d-EH250 (34) encodes nonactivatable toxin (7, 20, 50).
↵ b All Hnt and NM strains possessed fliC_H14.
↵ c One strain lost the stx₂ gene after isolation but before subtyping.

TABLE 2.

Comparison of non-stx virulence loci in STEC O91 isolates belonging to different serotypes

Gene^a	No. (%) of strains of STEC serotype:
Gene^a	O91:H8 (n = 1)	O91:H10 (n = 2)	O91:H14/Hnt/NM^b (n = 77)	O91:H21 (n = 20)
cdt ^c	0	0	0^d	14 (70.0)^d
subAB ^e	0	0	10 (13.0)	1 (5.0)
EHEC hlyA	1 (100.0)	0	55 (71.4)	19 (95.0)
saa	1 (100.0)	0	10 (13.0)^f	20 (100.0)^f
iha	1 (100.0)	1 (50.0)	75 (97.4)	20 (100.0)
lpfA _O26	1 (100.0)	0	76 (98.7)	16 (80.0)
lpfA _O113	1 (100.0)	1 (50.0)	73 (94.8)	19 (95.0)
espI	0	1 (50.0)	10 (13.0)	0
espP	1 (100.0)^g	0	49 (63.6)^g^,ⁱ	1 (5.0)^h^,ⁱ
ter ^j	0	0	0	1 (5.0)

↵ a The following genes encode the indicated proteins or activity: cdt, cytolethal distending toxin (CDT) (21, 22); subAB, subtilase cytotoxin (32); EHEC hlyA, EHEC hemolysin (1); saa, STEC autoagglutinating adhesin (Saa) (31); iha, iron-regulated gene A homologue adhesin (Iha) (43); lpfA_O26 and lpfA_O113, major subunits of long polar fimbriae of STEC O26 and O113, respectively (12, 46); espI, serine protease EspI (39); espP, serine protease EspP (10); and ter cluster, tellurite resistance.
↵ b All Hnt and NM strains possessed fliC_H14.
↵ c All presently known E. coli cdt alleles (cdt-I, cdt-II, cdt-III, cdt-IV, and cdt-V) were tested (4); all STEC O91:H21 isolates contained the cdt-V cluster (21).
↵ d STEC O91:H21 versus STEC O91:H14/Hnt/NM [fliC_H14]: P < 0.0001, Yates' corrected χ² = 45.35; CI₉₅ = 59.9 to 1,227.0.
↵ e Genes encoding both the A and B subunits of the subtilase cytotoxin were present.
↵ f STEC O91:H21 versus STEC O91:H14/Hnt/NM [fliC_H14]: P < 0.0001; χ² = 30.31; CI₉₅ = 7.65 to 18.24.
↵ g All strains possessed espPε.
↵ h espPβ.
↵ i STEC O91:H21 versus STEC O91:H14/Hnt/NM [fliC_H14]: P < 0.0002; Yates' corrected χ² = 13.48; CI₉₅ = 2.6 to 349.7.
↵ j All ter genes (terZABCDEF) were absent in all ter-negative strains, and all were present in the ter-positive strain.