Characterization of stx2 and its variants in Escherichia coli O157:H7 isolated from patients and animals

In this study, we investigated 72 Escherichia coli O157:H7 strains from humans and animals to determine stx2 and its variants by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Most isolates were found to carry stx2 or its variants and the stx2c was the dominant subtype; for the prevalence of stx2c in stx2-positive isolates was 89.6% (42/47). All stx2 and stx2c harboring isolates obtained from humans had caused diarrhea or hemolytic-uremic syndrome. Three strains isolated from a piglet and two cattle carried the stx2c gene with an IS1203v inserted sequence. Sequencing of this region revealed that, this 1.3 kb insertion was very similar to a previously identified IS 1203v sequence and the insertion interrupted the carboxyl end of the B subunit coding region of stx2c gene. The corresponding positions in the stx2c gene sequence in which the IS 1203v sequence was inserted was varied. The isolates possessing IS1203v were inactive in the Vero cell toxicity assay.

There are many serotypes of STEC strains, but the one most associated with the epidemic cases is O157:H7.The first major outbreak of STEC O157:H7 infection was reported in the United States in 1982 and was linked to eating undercooked ground beef from a fast-food restaurant chain (Riley et al., 1983).Since then, several outbreaks have been reported worldwide, including a large outbreak in Jiangsu and Anhui of Chinese Mainland during 1999 to 2000, which caused more than 200 deaths (Zheng et al., 2005).The pathogenesis of STEC O157:H7 infection in humans depends on many bacterial virulence factors including stx1, stx2, enterohemorrhagic E. coli (EHEC)-hemolysin, intimin and so on (Wang et al., 2008).But the most important factor is stx2 (Osawa et al., 2000;Kawano et al., 2008).The STEC O157:H7 strains have been reported to produce stx 1 , stx 2, and stx 2c (Schmitt et al., 1991;Tyler et al., 1991;Osawa et al., 2000;Zheng et al., 2005;Kawano et al., 2008), while other stx 2 variants were often observed from non-O157:H7 STEC.However, the stx 2 gene can be interruptted by a 1310 bp insertion sequence IS1203 variant (IS1203v) which is inserted in the region encoding the amino-terminus of the B subunit with a duplication of 3 bp at the target site and is not cytotoxic to Vero cells (Kusumoto et al., 1999;Jinneman et al., 2000).
Cattle are considered to be the major reservoir of E. coli O157:H7 (Baker et al., 2007;Wang et al., 2008;Williams et al., 2008).Recent studies have also demonstrated that, conventional pigs were permissive host for E. coli O157:H7 (Booher et al., 2002;Cornick and Helgerson, 2004), though the prevalence of the organism in these studies was generally low, except for the results from Chile and Chinese Mainland and both suggested that, pig might be an important source of this organism in some countries (Borie et al., 1997;Zheng et al., 2005).Also, one family outbreak had been specifically traced back to pork salami and the E. coli O157 isolates from the couple and the salami carried stx 1 , stx 2 and eae genes and shared the same PFGE pattern (Conedera et al., 2007).
In this paper, we analyzed the stx 2 produced by STEC O157:H7 strains isolated from both patients and animals, including cattle, pigs, goats, chicken.While polymerase chain reaction (PCR) analyzing of isolates produced bands of the predicted size for stx 2 , three isolates produced abnormally stx 2 gene amplification product containing a 1.3-kb insertion sequence IS1203v.This study describes PCR and Vero cell toxicity tests and sequencing of the insertion to fully understand this anomaly.

Bacterial strains
A total of 72 E. coli O157:H7 strains were isolated during several studies performed in Jiangsu Province from 1999 to 2002 and Chongqing Municipality in 2005 of the People's Republic of China (Table 1).The strains were isolated from patients' (two patients with HUS and 6 with diarrhea from Jiangsu), cattle (twenty-one from Jiangsu and 6 from Chongqing.),goats (twenty-three from Jiangsu); pig (Three from Jiangsu and 1 from Chongqing) and chicken feces (Nine from Jiangsu) as well as fresh mutton (One from Jiangsu).Their O and H antigens were further tested with anti-E.coli O157 and anti-E.coli H7 sera by slide agglutination test (SAT).Molecular identification of the O157 lipopolysaccharide antigen (rfbE) and H7 flagella antigen (fliC) genes were performed as described previously (Gannon et al., 1997;Desmarchelier et al., 1998).
The E. coli O157:H7 reference strain EDL933 was used as positive control for PCR detection of virulent marker and possessed stx1, stx2, eaeA, ehxA, EspA and Tccp as we have described previously (Wang et al., 2008).E. coli DH5α was stored in our laboratory.

Detection of stx2 and its variants by PCR and PCR-RFLP
The template DNA was prepared from a pure culture of isolates, grown in LB broth for 16 h at 37°C.Three ml of the culture were centrifuged at 8 000 rpm for 5 min and the pellet was re-suspended in 0.2 ml of ddH2O.The suspension was heated at 100°C for 10 min and then, centrifuged at 12 000 rpm for 5 min.The supernatant was used for the PCR template.E. coli O157:H7 isolates were examined by PCR assay to determine the presence of stx2 and its variants.PCR was performed in a total volume of 20 μl containing 0.5 unit of PrimeSTAR® HS DNA polymerase (Takara, Japan), 25 pmol of appropriate primers, 1.6 µl of dNTPs(2.5 mmol/L each), 4 µl 5×buffer(Mg 2+ plus)and 0.4 µl of the DNA template.The reaction was carried out in a Bio-Rad PCR system PTC-100 Peltier thermal cycler.
Primers used for the detection of stx2 gene and its variants are shown in Table 2.The stx2 genotypes harbored by the E. coli O157:H7 isolates were examined by PCR assay as described previously (Tyler et al., 1991;De Baets et al., 2004).The B subunit genes of strains positive with primers VT2-c and VT2-d-specific for stx2 and its stx2c variants were sub-typed by Tyler's PCRrestriction fragment length polymorphism (RFLP) method, which identifies stx2, stx2vha, and stx2vhb (Tyler et al., 1991).Amplicons were digested by enzymes HaeIII to discriminate stx2 from stx2c subtype genes.Use of restriction enzymes NciI and RsaI for PCR-RFLP analysis enabled classification of stx2c genes as stx2vha and stx2vhb.Primers VT2v-1 and VT2v-2 were also used to discriminate stx2c from stx2 subtype genes (Tyler et al., 1991).
Primers stx2D-1 and stx2D-2 (Table 2) were used for amplification of the initial 210 bp region of the stx2 B subunit of the BRY24 strain.The 210 bp region cycling conditions were as follows: Initial denaturation at 98°C for 5 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 10 s and extension for 20 s at 72°C; and final extension at 72°C for 6 min.Primers stx2D-3 and stx2D-4 (Table 2) set for the stx2 gene of 00F077 and 00F078 strains amplified a 1225 bp fragment from a region which spanned B subunit.The 1225 bp fragment cycling conditions were as follows: Initial denaturation at 98°C for 5 min; 30 cycles of denaturation at 98°C for 20 s, annealing at 48°C for 20 s and extension for 1 min 15 s at 72°C and final extension at 72°C for 6 min.PCR amplicons were run on a 2.5% agarose gel, stained with ethidium bromide and visualized under UV illumination.

DNA sequencing
The resulting PCR products of BRY24 and 00F078 with stx2-F and stx2-R primers were gel purified and ligated into pMD19-T simple vector, designating pMD19-BRY24 and pMD19-00F078, respecttively.Their constructed plasmids were sent to Takara Company for sequencing.DNA sequence information was analyzed using DNAssist software.

Vero cell cytotoxicity assay
Vero cell cytotoxicity assay was performed as described previously with minor modification (Teel et al., 2002).Isolates used were listed in Table 3. E. coli DH5α, a nontoxic laboratory strain, was used as the negative control.Briefly, Vero cells suspended in tissue culture medium (DMEM containing 10% fetal bovine serum) were seeded into wells (approximately10 4 cells/well) of a 96-well microtiter plate, leaving the exterior rows empty and incubated for 24 h at 37°C in the presence of 5% CO2.The tissue culture medium was removed by aspiration and replaced with 100 µl fresh medium.On the other hand, a single colony of each bacterial strain was removed, inoculated into 5 ml LB broth and shaken overnight at 37°C.After the cell concentration was adjusted to 1 x 10 9 CFU/ml, bacterial cultures were centrifuged and filtered with a 0.22 µm pore size filter.Filter-sterilized bacterial culture supernatants (100 µl) were then added to the first row of wells.Serial dilutions (1:5) of each supernatant were prepared in 96-well microtiter plates using tissue culture medium.The last row was not inoculated and served as a control for unintoxicated cell background.After incubation for 48 h, the cells were fixed in buffered formalin and stained with crystal violet.The intensity of the color of the fixed and stained cells was measured with a Bio-Rad microplate reader at 570 nm (A570).The staining intensity was proportional to the number of viable, attached tissue culture cells present before they were fixed to the well.The 50% cytotoxic dose (CD50) was the amount of toxin required to kill 50% of the cells in a well.The CD50 for each bacterial strain was determined by plotting the optical density at 570 nm of each dilution well after subtraction of the optical density at 570 nm for the blank against the log-transformed toxin dilution using Originpro7.5 software (OriginLab, Northampton, MA).

Nucleotide sequence accession number
The nucleotide sequences of the stx2 variant genes of STEC O157:H7 strain BRY24 and 00F078 have been submitted to the Genbank database under accession numbers GU983682 and GU983683.

RESULTS
Stx 2 genotypes among E. coli O157:H7 isolates from human and livestock sources E. coli O157:H7 isolates (n =72) were isolated from a variety of human and livestock sources.Forty eight iso-lates possessed stx 2 or its variants (Table 1).One isolate from cattle in Chongqing can amplify a 2740 bp fragment by using stx2-F and stx2-R primer pairs and a total of 47 (72.3%) isolates from Jiangsu Province had stx 2 or its variants.By using primers VT2-c and VT2-d, stx 2 can be detected in 45 isolates.Negative PCRs were repeated for confirmation, along with positive control.The PCR-RFLP and PCR with primers VT2v-1 and VT2v-2 of subtypes was used to distinguish stx 2, , stx 2vha , and stx 2vhb .The stx 2 was dominantly present in patients (3 of 5, 60%), only two stx 2 -positive isolates can be detected in animal.Stx 2vha were the dominant subtype in E. coli O157:H7 strains from animals; the prevalence of stx 2vha in stx 2positive isolates was 83.3% (40/48).Stx 2vhb variant was not found in any isolate (Figure 1).
Using stx 2 gene primers, stx2-F and stx2-R, a sequence of about 2 740 bp fragment was amplified in strains BRY24 and 00F078, which was larger than the normal amplified nucleotide sequence of stx 2 (1 400 bp).Then, the nucleotides fragments were sequenced by ligating into pMD19-T simple vector and the DNA sequence information was analyzed by using several bioinformatics software.The nucleotides sequences of subunit A of stx 2 BRY24 and stx 2 00F078 were identical and 99.1% homologous to stx 2 EDL933 .According to the nucleotide sequences of the B subunit of stx 2 BRY24 and stx 2 00F078 , two pairs of primers were designed.With primers stx2D-1 and stx2D-2, 210 bp fragment was obtained from strain BRY24, 254 bp fragment was obtained from strains 00F077 and 00F078.As the same, with primers stx2D-3 and stx2D-4, 1181 bp fragment was obtained from strain BRY24, and 1225 bp fragment was obtained from strains 00F077 and 00F078 (Figure 2).However, no amplicon was amplified in other isolates with these two pairs of primers.The amplicon of 00F077, amplified with primers stx2D-3 and stx2D-4, was sequenced by ligating into pMD19-T simple vector and the sequenced nucleotide was identical to the sequence of stx 2 00F078 .

Analysis of insertion sequence in BRY24, 00F077 and 00F078
The nucleotide sequences of the stx 2 genes of three E. coli O157:H7 were identical to the stx 2c gene sequence described by some researchers (Kusumoto et al., 1999;Lin et al., 1993;Schmitt et al., 1991;Teel et al., 2002), although, they were interrupted by a 1310 bp insertion sequence.In all three isolates, the IS were in the reverse orientation within the stx 2c gene.Both 1310 bp insertion sequences in 00F077 and 00F078 were identical to IS 1203v (Kusumoto et al., 1999).However, the 1310 bp sequence was most similar to IS 1203v with only a 2 bp substitution in the insertion sequence region of RBY24 when compared with the same region from the AB017524 sequence ("A" residue at position 581 in the RBY24 sequence and a "G" at the corresponding position 642 in sequence AB017524; "G" residue at position 587 in the RBY24 sequence and a "A" at the corresponding position 648 in sequence AB017524).However, the start codon of IS 1203v ORFb in 00F078 was mutated to "ATA" in BRY24 and the length of ORFb in BRY24 was 3 bp shorter than AB017524's (Figure 3).The corresponding positions in the stx 2c gene sequence at which the IS 1203v sequence were located were 212 in the B subunit for the isolates 00F077 and 00F078, and 168 in the B subunit for RBY24.
For 00F077, 00F078 and BRY24 IS1203v sequences, the three nucleotides ("ATT", "ATT" and "AAC", respecttively) preceding the insertion site were repeated after IS 1203v followed by the remainder of the stx 2c gene sequence for the regions sequenced.IS 1203v in all three isolates were inserted in the region encoding the amino-terminus of the B subunit.

Cytotoxicity
The Vero cell cytotoxicity of selected isolates was analyzed by using a microtiter plate cytotoxicity assay.The supernatants of CYB42 and DH5α were not cytotoxic to Vero cells as expected.On the contrary, the supernatants of EDL933 (Containing stx1 and stx2) and 00B015 (Containing stx2c but not containing stx1) isolates were cytotoxic to Vero cells as expected.Nevertheless, BRY24, 00F077 and 00F078 were not cytotoxic to Vero cells as expected (Jinneman et al., 2000).

DISCUSSION
In human, Shiga toxins are the major virulence factors of STEC responsible for HC and HUS.The association of STEC with HC and HUS implies that stx 2 is more closely to these diseases than stx 1 (Miceli et al., 1999;Siegler et al., 2003).Recent studies showed that, the strains carrying stx 2vha might be less virulent and less frequently cause bloody diarrhea (Zheng et al., 2005;Kawano et al., 2008).Therefore, stx 2 sub-typing is suggested to be helpful in understanding the role of the different subtypes in clinical medicine and epidemiology.In this study, we  (Kusumoto et al., 1999).However, the start codon of IS 1203v ORFb in 00F078 was mutated to "ATA" in BRY24, and the length of IS 1203v ORFb in BRY24 was 3 bp shorter than AB017524's (Kusumoto et al., 1999).The solid lines represent the stop of IS 1203v ORFa and the dash lines represent the start of IS 1203v ORFb.
analyzed a collection of 72 E. coli O157:H7 isolates recovered from cattle, goat, pig, chicken and human patients to further characterize the stx 2 that they contained.Two main stx 2 subtypes were detected (stx 2 and stx 2c ) and no combination of these two subtypes was detected.Two pairs of primers (stx2-F and stx2-R, VT2-c and VT2-d) can amplify stx 2 and stx 2c , though the amplicons amplified by primers VT2-c and VT2-d can be further subtyped by using Tyler's PCR-RFLP method (Piérard et al., 1997;Piérard et al., 1998).Stx 2 genotype isolates caused more severe symptoms (two with HUS and 1 with bloody diarrhea) than stx 2c harboring isolates (three with watery diarrhea), though the case load was shortage.However, Friedlich et al. (2002) have shown that stx 2c is the only stx 2 variant associated with HUS, even the risk of developing HUS was significantly lower after infection with stx 2 -bearing.Therefore, E. coli O157:H7 harboring stx 2c may be a threaten pathogen and become the dominant variant in the outbreak.Nevertheless, two isolates that did not contain stx 2 can also cause clinical symptoms, such as watery diarrhea.It may suggest that accessory virulence factors can be present in some fully pathogenic strains, such as intimin, EHEC hemolysin, and tccp (Wang et al., 2008).
Compared to other STEC O157:H7, the identification of an insertion of approximately 1300 bp in the stx 2c gene for BRY24, 00F077 and 00F078 isolates was of particular concern to us.Kusumoto et al. (1999) reported that the stx 2vhd gene (In fact the stx 2c gene) (Lin et al., 1993), one of the stx 2 variants was interrupted by IS1203v, whose target site was 33 bp away at the amino terminal end of the coding region for the B subunit, resulting in the inactivation of the stx 2 gene.Based on the related nucleotide sequences data, the IS 1203v element in stx 2c of 00F077 or 00F078 was inserted nearer the carboxyl terminal end of the B subunit coding region than BRY24's.IS 1203 belongs to the IS 3 family which are between 1200 and 1550 bp with inverted terminal repeats and containing two consecutive and partially overlapping open reading frames (ORF) (Mahillon and Chandler, 1998).IS 1203 is flanked by 26 bp imperfect terminal repeats and two ORFs, with the second ORF being previously identified as encoding a transposase (Paton and Paton, 1994).The E. coli O157:H7 plasmid sequences include IS 1203, the sequence of which has been reported in other STEC strains including preceding the stx 1 gene in a E. coli O111:H-strain (Paton et al., 1993).IS 1203 has been reported to interrupt the espP gene of a non-sorbitol fermenting E. coli O157:H7 strain (Brunder et al., 1999).Moreover, other researches indicated that, IS1203v was inserted into the stx 2 gene of E. coli O157:H7 strains at different sites (Jinneman et al., 2000;Okitsu et al., 2001).Interestingly, two O157:H7 strains isolated from humans carrying the stx1 gene with an IS 1203 that was inserted into the coding region of the A subunit (Suzuki et al., 2004).In this study, the result was similar to a report from Chinese Mainland, in which E. coli O157:H7 possessing IS 1203v inserted stx 2c gene was prevalent (Zheng et al., 2005).Moreover, the 00F077 and 00F078 isolates were obtained from cattle in the same farm and their stx 2c gene had the identical sequence and that might suggest a common origin.
In conclusion, based on the analysis of stx 2 genotype and clinical manifestations caused by E. coli O157:H7, it might be confirmed that, stx 2 genotype was one of the important risky factors of disease severity and that, stx 2c was the dominant genotype of STEC O157:H7 isolated from domestic animals.Three isolates, obtained from a piglet and two cattle, have acquired an IS 1203v within the stx 2c gene and did not produce a functional stx2c protein.

Figure 3 .
Figure3.Nucleotide sequences comparison of IS1203v ORFb in the stx2c for both BRY24 and 00F078 strains.The 1310 bp insertion sequence in 00F078 was identical to IS 1203v(Kusumoto et al., 1999).However, the start codon of IS 1203v ORFb in 00F078 was mutated to "ATA" in BRY24, and the length of IS 1203v ORFb in BRY24 was 3 bp shorter than AB017524's(Kusumoto et al., 1999).The solid lines represent the stop of IS 1203v ORFa and the dash lines represent the start of IS 1203v ORFb.

Table 1 .
The stx2 genotype of E. coli O157:H7 isolates from Jiangsu and Chongqing, People's Republic of China.
b 1 a Stx2 or its variants was not detected; b containing IS 1203v.

Table 2 .
Primers used for detection and typing stx2 variants.