J Bacteriol Virol.  2009 Jun;39(2):79-95. 10.4167/jbv.2009.39.2.79.

Molecular Genetic Characterization of Shiga Toxin-producing E. coli Isolated from Diarrhea Patients and Cattle in Gwangju Area, Korea

• Kim MJ
• Kim SH
• Kim TS
• Kee HY
• Seo JJ
• Kim ES
• Park JT
• Chung JK
• Lee BK
• Lee J

• KMID: 1474168
• DOI: http://doi.org/10.4167/jbv.2009.39.2.79

Abstract

Shiga toxin-producing Escherichia coli (STEC) can cause a broad spectrum of human illness ranging from symptom-free to hemolytic uremic syndrom (HUS). Associations between known or putative virulence factors of STEC and diseases in human were investigated. PCR analyses showed that 33 (78.6%) isolates carried an ehxA enterohemolysin gene and 6 (14.3%) isolates possessed an saa autoaggutinating adhesin gene, and 31 (73.8%) isolates carried an eae intimin gene (7 isolates with type beta, 16 with type gamma, and 3 with type epsilon). Twenty-nine (69%) isolates from patients carried eae+, ehxA+, saa- (genotype A) and 68 (86%) isolates from asymptomatic outbreaks and 4 (36%) isolates from bovine possessed eae-, ehxA+, saa+ (genotype C). Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. In HEp-2 cell adherence assay, isolates carrying eae gene exhibited a localized adherence phenotype, the other isolates carrying saa showed LC (loose clusters of bacteria) and IS (isolated bacteria). In conclusion, most STEC isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. But these results may be useful information for pathogenesis judgement of STEC.

Keyword

Shiga toxin-producing Escherichia coli; saa; ehxAx; eae; HEp-2 cell adherence assay

MeSH Terms

Animals
Cattle
Diarrhea
Disease Outbreaks
Enteropathogenic Escherichia coli
Escherichia coli Proteins
Feces
Hemolysin Proteins
Humans
Korea
Lifting
Molecular Biology
Phenotype
Plasmids
Polymerase Chain Reaction
Shiga-Toxigenic Escherichia coli
Virulence Factors
Escherichia coli Proteins
Hemolysin Proteins
Virulence Factors

Figure

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  Figure 1. The procedure of HEp-2 cell adherence assay.

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  Figure 2. Distribution of the STEC genotype by multiplex PCR. A: eaeA+, ehxA+, saa-, B: eaeA+, ehxA-, saa-, C: eaeA-, ehxA+, saa+, D: eaeA-, ehxA+, saa-, E: eaeA-, ehxA-, saa+, F: eaeA-, ehxA-, saa- (+: gene present; -: gene absent).

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  Figure 3. Multiplex PCR analysis of the saa (119 bp), eae (384 bp), the ehxA (534 bp) genes. M: 100 bp ladder, lane 1: genotype A (isolate GJ-04-08-23), lane 2: genotype C (isolate Jinwol-SBM), lane 3: genotype D (isolate bovine 20), lane 4: genotype A (isolate GJ-08-07-200), lane 5: genotyep F (isolate bovine 31), lane 6: negative control, lane 7: positive control (EDL 933).

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  Figure 4. PCR result for the detection of eaeβ (520 bp), eaeγ (778 bp) and eaeε (2,069 bp) genes. M: 100 bp ladder, lane 1: eaeβ + (isolate GJ-04-07-84), lane 2: eaeγ + (isolate GJ-05-06-290), lane 3: eaeε + (isolate GJ-05-09-32), lane 4: positive control (EDL 933), lane 5: negative control.

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  Figure 5. PCR result for the detection of espA (299 bp), espB (633 bp), espD (939 bp) and tir (1,550 bp) genes. M: 100 bp ladder, lane 1~4: espA +, espB +, espD +, tir + (isolate GJ-05-09-032), lane 5: negative control, lane 6~9: positive control (EDL 933).

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  Figure 6. PCR analysis of the espP (301 bp)genes. M: 100 bp ladder, lane 1: espP + (isolate GJ-07-06-324), lane 2: negative control, lane 3: positive control (EDL 933).

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  Figure 7. Multiplex PCR to distinguish an intact selC locus from one disrupted by LEE. M: 100 bp ladder, lane 1: disrupted selC (418 bp, isolate GJ-05-06-150), lane 2: Intact selC (527 bp, isolate GJ-05-06-290), lane 3: ND (not detected).

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  Figure 8. Adherence patterns of STEC in HEp-2 cell culture. A: LA pattern, isolate GJ-04-07-84, B: LC pattern, isolate Jinwol 9, C: IS pattern: isolate Jinwol SBM, D: DA pattern, isolate bovine 27, E: AA pattern, isolate GJ-08-07-54, F: Negative control.

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