Chinese Medical Journal 2002;115(6):815-818
Identification of Shiga- like toxin Escherichia coli isolated from children wi th diarrhea by polymerase chain reaction

ZHU Qingyi 朱庆义 ,  LI Lianqing 李连青,  GUO Zhaobiao 郭兆彪,  YANG Ruifu 杨瑞馥

ZHU Qingyi 朱庆义 (Laboratory of Molecular Microbiology, Shanxi Children’s Hospital, Taiyuan 030013, China)

LI Lianqing 李连青 (Laboratory of Molecular Microbiology, Shanxi Children’s Hospital, Taiyuan 030013, China)

GUO Zhaobiao 郭兆彪 (Institute of Microbiology and Epidemiology, Chinese Academy of Military Medical Sciences, Beijing 100071, China)

YANG Ruifu 杨瑞馥 (Epidemiology, Chinese Academy of Military Medical Sciences, Beijing 100071, China)

Correspondence to:, (Tel: . Fax:. E-mail:)
Keywords
Shiga- like toxin E. coli;diarrhea;polymerase chain reaction
Abstract
Objective To evaluate the etiology of Shiga- like toxin- producing Escherichia coli (SLTEC) in children with diarrhea.
Methods We designed and synthesized 3 pairs of primers located in the SLT1, SLT2, and eaeA genes of enterohaemorrhagic Escherichia coli(EHEC), while the virulent genes SLT1, SLT2, and eaeA from E. coli species were amplified by polymerase chain reaction (PCR).
Results One strain of EHEC with SLT1, SLT2, and eaeA in 29 reference strains of diarrhea- causing E. coli(DCEC) and 10 strains of other enterobacteria detected by PCR had positive reactions, while all other DCEC and enterobacteria were negative. Of 474 strains of E. coli isolated from 1032 children with diarrhea and detected by PCR, 20 strains of SLT1 producing E. coli(4. 2%) positive, and 7 strains of SLT2 producing E. coli(1. 5%) positive; while of 74 strains of entero- SLTs- producing and invasive Escherichia coli(ESIEC), 15 strains of SLT1 (20. 3%) and 5 strains of SLT2 (6. 8%) were positive.
Conclusion
Shiga- like toxin E. coli has been identified as a major etiologic agent of
children with diarrhea in Taiyuan, China.

Toxigenic Escherichia coli strains that cause diarrhea in human s and domestic animals have been classified into three categories: enterotoxigen ic E.coli (ETEC), Shiga- like toxin E. coli (SLTEC), and necrotoxigenic E. coli (NTEC).[1- 3] The ETEC strains may synthesize thermolabile (LT) and/or thermostable (ST) enterotoxins and cause diarrhea in children. Th e SLTEC strains may produce three main types of Shiga- like- toxin (SLT1, SLT2, and SLT2v) that are similar to the Shiga toxin synthesized by Shigella dysenteri ae type Ⅰ (such as O157∶H7). SLTEC (SLT1 and/or SLT2) strains are recogni zed as a cause of hemorrhagic colitis and the hemolytic- uremic syndrome in humans. Furthermore, SLTEC strains producing SLT2v are responsible for edema in pigs. [4,5]In a recent discovery, a new category of the entero- SLTs- produc ing and invasive Escherichia coli (ESIEC) was isolated from patients with di arrhea in China.[6] In this study, we tested the SLTEC isolated from children with diarrhea in Taiyuan for the SLTs genotypes by using the polymerase chain r eaction (PCR).

METHODS Bacterial strains
The bacterial strains used in this study were reference strains of enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli, Shigella, Salmonella, Kl. pneumonia, P. morganii, and Ps. aeruginosa (Table 1) . They were provided by the National Institute for Control of Pharmaceutical and Biological Products, and G. K. Morris (The Centers for Disease Control in the United States). A total of 32 strains of ETEC, 8 strains of enteropathogenic Escherichia coli (EPEC), and 2 strains of entero- invasive E. coli (EIEC) were isolated from children with diarrhea in Shanxi Children’s Hospital during 1984-1986. The serotypes and virulence factor of these bacterial strains were identified by animal test, PCR, and oligonucleotide probe hybridization, respectively.

Bacterial isolates and identification in fecal specimens
A total of 1032 stool specimens from children with diarrhea, both outpatients an d inpatients, were submitted to the Shanxi Children’s Hospital from January to D ecember 1997 less than 3 days after admission. Fresh stool specimens were place d in cleaning penicillin bottles upon collection in the laboratory. All of stoo l specimens were processed for routine enteric pathogen culture including MacCok ey agar and Sorbitol- MacCokey agar. Following overnight incubation, five suspi cious colonies from each plate were tested for serotypes and virulence (antiseru m of EPEC, EIEC, and EHEC (O157∶H7) provided by the Lanzhou Vaccine and Serum I nstitute and the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. For detection of the LT and ST of ETEC, we used SPA- coagg lutination and PCR assay as described in detail previously.[7,8]

PCR amplification
Synthetic oligonucleotide

Oligonucleotide used as primers in PCR was designed on the basis of the DNA pol ymerase common type gene sequence for SLT1, SLT2, and eaeA of EHEC O157∶H7. Th e sequences of the SLT1 primers were 5’- TTTA- CCTTAGACTTCTCGAC- 3’ and 5’- CACCAGACAATGT- AACCGCTG- 3’, amplifying a 325 bp DNA fragment. The amplifica tion product was cleaved by Hinf Ⅰ into two restriction fragments (37 bp and 288 bp). The sequences of the SLT2 primers were 5’- TTTACGATAGACTTTTCGAC- 3’ and 5’- GCGTCAT- CGTATCACAAGAAC- 3’, amplifying a 571 bp DNA fragment. The amplification product was cleaved by Hea Ⅲ into two restriction fragments (228 bp and 343 bp). The sequences of the eaeA primers were 5’- CAGGTCGTCGTGT- CTGCT AAA- 3’ and 5’- ACAGCGTGGTTGGATCAACCT- 3’,amplifying a 1089 bp DNA fragment. The amplification product was cleaved by Hinf Ⅰ into two restriction fragments (730 bp and 359 bp). All oligonucleotides were synthesized on an applied b iosystems synthesizer with the automated phosphoramidate coupling method (primer s given by the Institute of Microbiology, Chinese Academy of Sciences). DNA extraction A half- loopful of bacteria growth was removed from a MacCokey- sorbitol agar pl ate, suspended in 100 μl of sterile saline, given 100 μl of 2×PCR buffer [ 1×PCR buffer: 10 mmol/L Tris- HCl (pH 8. 3), 50 mmol/L KCl, 2. 0 mmol/L MgCl 2, 0. 05% gelatin, 0. 5% Triton X- 100, 0. 5% Tween- 20], heated at 100℃ for 1 0 min, and centrifuged at 13 000 r/min for 10 min. 15 μl of supernatant was taken for PCR amplification.

PCR assay
Amplification was done in a 10 μl reaction mixture consisting of 0. 2 μmol/L o f each primer, 200 μmol/L of each deoxynucleoside triphosphate (dNTP, Promega) , 1 U Taq DNA polymerase (Promega), 1×PCR buffer and 15 μl of DNA extracts. The mixtures were overlaid with 1 drop of mineral oil. PCR was performed at 93 ℃ for 3 min and then 30 cycles of denaturation at 93℃ for 1 min, annealing at 58℃ for 1 min, and elongation at 72℃ for 1 min, with 5 min of extension at 72 ℃ after 30 cycles in a DNA thermal cycler (PTC- 100, MJ Research Inc. ). Each PCR assay included the reference strain O157∶H7 as a positive control and water as a negative control. Amplified products were electrophoretically analy zed on 1. 5% agarose gels and stained with ethidium bromide. A positive result was included by the presence of the 325 bp, 571 bp, and 1089 bp amplified D NA fragment for SLT1, SLT2 and eaeA gene revealed by electrophoresis on a 1. 5% agarose gel.

Digestion of PCR products
Restriction endonucleases Hinf Ⅰ and Hea Ⅲ were used to digest amplified fragments obtained from isolation extracts. After amplification in a 15 μl reaction, DNA was precipitated in 100 μl ethanol with 0. 3 mol/L NaAc, and then resuspended in 5 μl of sterile water. Five microliters were digested with 10 U Hif I, Hae Ⅲ, and Hif Ⅰ for SLT1, SLT2, and eaeA, respectively, in a final volume of 10 μl. Digest fragments were electrophoretically analyzed on 1. 5% agarose gels and stained with ethidium bromide (Table 2 and Fig. ). Table 2. Results of the detection of SLTs typing of E. coli isolated from children with diarrhea

RESULTS

Specificity of primers for detecting genes for SLTs
Thirty- nine reference strains of enteropathogenic bacteria and 42 strains of diarrhea- causing E. coli (ETEC, EPEC, EIEC) isolated from children with diarrhea in Shanxi Children’s Hospital during 1984-1986 were tested for amplification to DNA of SLT virulence factors by PCR. Among 39 reference strains of enteropathogenic bacteria and 42 strains of DCEC, only one strain of EHEC (O157∶H7) was positive for the SLT1, SLT2, and eaeA genes, whereas all of the EPEC, ETEC, EIEC, and other enteropathogenic bacteria were negative for the SLT1, SLT2, and eaeA genes. The oligonucleotide derived from the genes for SLTs amplified only to EHEC or SLTEC. The amplification products of SLTs were analyzed by endonuclease- cleavage as shown in Fig. . The results indicated that the primers for detecting genes for SLTs had a high specificity by PCR.

Results of DCEC detection in fecal specimens
A total of 625 E. coli strains were isolated from 1032 fecal specimens from children with diarrhea in Shanxi Children’s Hospital from January to December 1997. Two hundred and five strains (32. 8%) of various DCEC of the 625 E. coli strains were identified by serogroups and enterotoxin, these strains belonged to ETEC (12. 4%), EPEC (3. 1%), EIEC (0. 5%), ESIEC (11. 4%), and SLTEC (4. 1%). The results are shown in Table 3, indicating that DCEC are important pathogenic agents of children with diarrhea in Taiyuan.

Detection of SLT typing of E. coli isolated from children with diarr hea
All of the 625 strains of E. coli and DCEC isolated from children with diarr hea were detected by PCR for SLT virulence genes (Table 2). Among the 474 E. coli strains, 20 strains (4. 2%) were SLT1 positive, and 7 strains (1. 5%) o f E. coli were SLT2 positive. Of 74 ESIEC strains, 15 (20. 3%) were SLT1 pos itive, 5 (6. 8%) were SLT2 positive, while all of the other DCEC were SLTs nega tive.

DISCUSSION

Shiga- like toxin producing E. coli is a major etiologic agent of intraintes tinal and outintestinal tract infections in humans and domestic animals. They h ave been classified into three categories: EHEC (O157∶H7),verocytotoxin E. c oli (VTEC), and ESIEC.[1- 3,5,6] The SLTEC strains may produce three mai n types of Shiga- like toxins (SLT1, SLT2, and SLT2v), which are similar to the Shiga toxin synthesized by Shigella dysenteriae type 1. EHEC and VTEC producing the eaeA virulence factor are a cause of hemorrhagic colitis and hemolytic urem ic syndrome.[3,5]An important difference between SLAEC, EHEC, and ES IE C strains may be the tissue specificity of the plasmid- encoding adhecins (EF/ 1). ESIEC, unlike SLTEC and EHEC, is a pathogen of children with diarrhea, a nd d oes demonstrate to human tissue culture cells such as Hep- 2. EF/1 is critical virulence factor and protective antigen of ESIEC; it can adhere to Hep- 2 cells in aggregation, but does not hybridize with the EAggEC DNA probe, which was dev eloped specifically for entero- aggregative E. coli (EAggEC).[9- 12] A total of 625 E. coli strains isolated from children with diarrhea in Taiy uan were identified by serogroups and enterotoxin, and among the various DCEC in cluded were ESIEC, EPEC, ETEC, and EIEC. All of the E. coli strains were d etected by PCR for the SLT1, SLT2, and eaeA virulence genes.

Among the 474 E. coli str ains, 20 (4. 2%) were SLT1 positive, 7 (1. 5%) were SLT2 positive, but did not a mplify with the eaeA virulence gene, showing that the strains of SLTEC are unlik e EHEC and ESIEC.[13] Of the 74 ESIEC strains, 15 (20. 3%) were SLT1 posi t ive, and 5 (6. 8%) were SLT2 positive. These strains of SLTEC could aggregate w ith the specific antiserum of ESIEC, but did not hybridize with the EF/1 probe o r with the specific antiserum of EPEC and EIEC, because these SLIEC strains do not belong to EPEC and EIEC. Complementary to the plasmid of the invasive ipa H gene from Shigella and EIEC, this primer pair did not amplify DNA from Shigell a and EIEC, and did not aggregate with the specific antiserums of EPEC, E IEC, and SLTEC. Biological characteristics of the SLTEC strain are ferments la ctose slowly, indole (+), lysine (-), sorbitol (-), and KCN (-). The t ypical symptoms for SLTEC infection in children are diarrhea, abominal pain, fe ver, unform stool, without bloody purulent stool. Based on the clinical sympto ms observed in children with diarrhea, SLTEC infection is distinguishable from t hat of EHEC, EIEC, ETEC, ESIEC, and similar to VTEC. The results showed that S LTEC is a newly discovered DCEC for children with diarrhea in Taiyuan.

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