1 Scope
This standard specifies the examination method of diarrheagenic Escherichia coli in foods.
This standard applies to the examination of diarrheagenic Escherichia coli in foods.
2 Terms, Definitions and Abbreviations
2.1 Terms and definitions
For the purpose of this standard, the following terms and definitions apply.
2.1.1 Diarrheagenic Escherichia coli
A kind of Escherichia coli can cause diarrhea-based symptom in human body via contaminated food. The common diarrheagenic Escherichia coli mainly includes enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli (including enterohemorrhagic Escherichia coli) and enteroaggregative Escherichia coli.
2.1.2 Enteropathogenic Escherichia coli
The Escherichia coli that can cause adhesion and wiping damage of host intestinal mucosa epithelial cell and does not produce Shiga toxin. This bacterium is the main pathogenic bacteria causing infant diarrhea, with high infectivity, which can be fatal in severe cases.
2.1.3 Enteroinvasive Escherichia coli
The Escherichia coli that can invade intestinal epithelial cell to cause dysentery-like diarrhea. This bacterium is free from power, lysine decarboxylic reaction and lactose fermentation, while with both biochemical reaction and antigenic structure approximate to that of Shigella dysenteriae. The key genes intruding epithelial cell are antigen encoding gene and controlling gene of invasive plasmid, such as ipaH-gene and ipaR-gene (also referred to as invE-gene).
2.1.4 Enterotoxigenic Escherichia coli
The Escherichia coli that can secrete heat-stable enterotoxin or/and heat-labile enterotoxin. This bacterium may cause infant and tourist diarrhea, which shows mild water-like diarrhea generally, and may shows serious cholera-like symptom, with low fever or no fever. Diarrhea is often self-limiting and can be self-healing in 2d~3d generally.
2.1.5 Shiga toxin-producing Escherichia coli (Enterohemorrhagic Escherichia coli)
The Escherichia coli that can secrete Shiga toxin and cause adhesion and wiping damage of host intestinal mucosa epithelial cell. Some Shiga toxin-producing Escherichia coli can cause clinically hemorrhagic colitis (HC) and bloody diarrhea in human, and may be further developed to become hemolytic uremic syndrome (HUS), this kind of Shiga toxin-producing Escherichia coli refers to enterohemorrhagic Escherichia coli.
2.1.6 Enteroaggregative Escherichia coli
Enteroaggregative Escherichia coli does not invade intestinal epithelial cell, but can cause intestinal fluid accumulation. It neither produces heat-stable enterotoxin/heat-labile enterotoxin nor Shiga toxin. It is only characterized by enteroaggregative adhesion on Hep-2 cell, thus also referred to as Hep-2 cell adhesive Escherichia coli.
2.2 Abbreviations
For the purposes of this standard, the following abbreviations apply.
2.2.1 DEC: Diarrheagenic Escherichia coli
2.2.2 EPEC: Enteropathogenic Escherichia coli
2.2.3 EIEC: Enteroinvasive Escherichia coli
2.2.4 ETEC: Enterotoxigenic Escherichia coli
2.2.5 STEC: Shiga toxin-producing Escherichia coli
2.2.6 EHEC: Enterohemorrhagic Escherichia coli
2.2.7 EAEC: Enteroaggregative Escherichia coli
2.2.8 escV: gene encoding LEE-encoded type Ⅲ secretion system factor
2.2.9 eae: gene encoding intimin for Escherichia coli attaching and effacing
2.2.10 bfpB: bundle-forming pilus B;
2.2.11 stx1: Shiga toxin one
2.2.12 stx2: Shiga toxin two
2.2.13 lt: heat-labile enterotoxin
2.2.14 st: heat-stable enterotoxin
2.2.15 stp (stIa): heat-stable enterotoxins initially discovered in the isolates from pigs
2.2.16 sth (stIb): heat-stable enterotoxins initially discovered in the isolates from human
2.2.17 invE: invasive plasmid regulator
2.2.18 ipaH: invasive plasmid antigen H-gene
2.2.19 aggR: aggregative adhesive fimbriae regulator
2.2.20 uidA: β-glucuronidase gene
2.2.21 astA: enteroaggregative heat-stable enterotoxin A
2.2.22 pic: protein involved in intestinal colonization
2.2.23 LEE: Locus of enterocyte effacement
2.2.24 EAF: EPEC adhesive factor
3 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
3.1 Constant temperature incubator: 36℃±1℃,42℃±1℃.
3.2 Refrigerator: 2℃~5℃.
3.3 Thermostatic water bath: 50℃±1℃, 100℃ or adapting 1.5mL or 2.0mL metal bath (95℃~100℃).
3.4 Electronic balance: with sensibility of 0.1g and 0.01g.
3.5 Microscope: 10×~100×.
3.6 Homogenizer.
3.7 Oscillator.
3.8 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipettor and pipette tip.
3.9 Aseptic homogenizing cup or aseptic homogenizing bag: with capacity of 500mL.
3.10 Aseptic culture dish: 90mm in diameter.
3.11 pH meter or precision pH paper.
3.12 Microcentrifuge tube: 1.5mL or 2.0mL.
3.13 Inoculating loop: 1μL.
3.14 Low-temperature high-speed centrifuge: rotation speed ≥13000 r/min, temperature control at 4℃~8℃.
3.15 Microbial identification system.
3.16 PCR instrument.
3.17 Micropipettor and pipette tip:0.5μL~2μL, 2μL~20μL, 20μL~200μL, 200μL~1000μL.
3.18 Horizontal electrophoresis apparatus: including power supply, electrophoresis tank, glue-manufacturing tank (with length >10cm) and comb.
3.19 8-tube strip and 8–cover strip (flat cover/convex cover)
3.20 Gel-imaging apparatus.
4 Media and Reagents
4.1 Nutrient broth: see A.1.
4.2 Enterobacteria enrichment broth: see A.2.
4.3 Maconkey agar (MAC): see A.3.
4.4 Eosin methylene blue agar (EMB):see A.4.
4.5 Triple sugar iron (TSI)agar: see A.5.
4.6 Peptone water and indole reagent: see A.6.
4.7 Semi-solid agar: see A.7.
4.8 Urea agar (pH 7.2): see A.8.
4.9 Potassium cyanide(KCN) medium: see A.9.
4.10 Oxidase reagent: see A.10.
4.11 Gram stain solution: see A.11.
4.12 BHI broth: see A.12.
4.13 Formalin (including 38%~40% of formaldehyde).
4.14 Identification kit.
4.15 Escherichia coli diagnostic serum.
4.16 Aseptic deionized water.
4.17 0.85% aseptic normal saline.
4.18 TE(pH8.0): see A.13.
4.19 10×PCR reaction buffer: see A.14.
4.20 25mmol/L MgCl2.
4.21 dNTPs: dATP, dTTP, dGTP and dCTP with concentration of 2.5mmol/L.
4.22 Taq enzyme in 5U/L.
4.23 Primer.
4.24 50×TAE electrophoretic buffer: see A.15.
4.25 Agarose.
4.26 Ethidium bromide(EB) or other nucleic acid dye.
4.27 6×loading buffer solution: see A.16.
4.28 Marker: Molecular weight includes bands of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp and 1500bp.
4.29 Diarrheagenic Escherichia coli PCR kit.
5 Examination Procedures
See Figure 1 for examination procedures of diarrheagenic Escherichia coli.
Figure 1 Examination Procedures of Diarrheagenic Escherichia Coli
6 Operation Steps
6.1 Sample preparation
6.1.1 Solid or semi-solid sample
Foe solid or semi-solid sample, weigh 25g of examined sample by aseptic operation, add it into a homogenizing cup with 225mL of nutrient broth to homogenize by a spinning blade-type homogenizer at 8000r/min~10000r/min for 1min~2min; or add the examined sample into a homogenizing bag with 225mL of nutrient broth to homogenize by a slap-type homogenizer for 1min~2min.
6.1.2 Liquid sample
Weight 25mL of examined sample by aseptic technique, add it into an aseptic conical flask with 225mL of nutrient broth (proper amount of aseptic glass beads may be preset in the flask), and then oscillate the flask to mix them well.
6.2 Enrichment
Culture the homogeneous sample solution prepared in 6.1 at 36℃±1℃ for 6h. Take 10μL of the solution, inoculate it into 30mL enterobacteria enrichment broth tube, and then culture at 42℃±1℃ for 18h.
6.3 Isolation
Carry out streak inoculation for the enrichment broth into MAC and EMB agar plates, culture them at 36℃±1℃ for 18h~24h, and then observe colony characteristics. On the MAC agar plate, the typical colony decomposing lactose is brick red to peach while the colony not decomposing lactose is colorless or light pink. On the EMB agar plate, the typical colony decomposing lactose is purple black with or without metallic luster in center while the colony not decomposing lactose is colorless or light pink.
Foreword I
1 Scope
2 Terms, Definitions and Abbreviations
3 Apparatus and Materials
4 Media and Reagents
5 Examination Produces
6 Operation Steps
7 Result Report
Appendix A Media and Reagents
1 Scope
This standard specifies the examination method of diarrheagenic Escherichia coli in foods.
This standard applies to the examination of diarrheagenic Escherichia coli in foods.
2 Terms, Definitions and Abbreviations
2.1 Terms and definitions
For the purpose of this standard, the following terms and definitions apply.
2.1.1 Diarrheagenic Escherichia coli
A kind of Escherichia coli can cause diarrhea-based symptom in human body via contaminated food. The common diarrheagenic Escherichia coli mainly includes enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli (including enterohemorrhagic Escherichia coli) and enteroaggregative Escherichia coli.
2.1.2 Enteropathogenic Escherichia coli
The Escherichia coli that can cause adhesion and wiping damage of host intestinal mucosa epithelial cell and does not produce Shiga toxin. This bacterium is the main pathogenic bacteria causing infant diarrhea, with high infectivity, which can be fatal in severe cases.
2.1.3 Enteroinvasive Escherichia coli
The Escherichia coli that can invade intestinal epithelial cell to cause dysentery-like diarrhea. This bacterium is free from power, lysine decarboxylic reaction and lactose fermentation, while with both biochemical reaction and antigenic structure approximate to that of Shigella dysenteriae. The key genes intruding epithelial cell are antigen encoding gene and controlling gene of invasive plasmid, such as ipaH-gene and ipaR-gene (also referred to as invE-gene).
2.1.4 Enterotoxigenic Escherichia coli
The Escherichia coli that can secrete heat-stable enterotoxin or/and heat-labile enterotoxin. This bacterium may cause infant and tourist diarrhea, which shows mild water-like diarrhea generally, and may shows serious cholera-like symptom, with low fever or no fever. Diarrhea is often self-limiting and can be self-healing in 2d~3d generally.
2.1.5 Shiga toxin-producing Escherichia coli (Enterohemorrhagic Escherichia coli)
The Escherichia coli that can secrete Shiga toxin and cause adhesion and wiping damage of host intestinal mucosa epithelial cell. Some Shiga toxin-producing Escherichia coli can cause clinically hemorrhagic colitis (HC) and bloody diarrhea in human, and may be further developed to become hemolytic uremic syndrome (HUS), this kind of Shiga toxin-producing Escherichia coli refers to enterohemorrhagic Escherichia coli.
2.1.6 Enteroaggregative Escherichia coli
Enteroaggregative Escherichia coli does not invade intestinal epithelial cell, but can cause intestinal fluid accumulation. It neither produces heat-stable enterotoxin/heat-labile enterotoxin nor Shiga toxin. It is only characterized by enteroaggregative adhesion on Hep-2 cell, thus also referred to as Hep-2 cell adhesive Escherichia coli.
2.2 Abbreviations
For the purposes of this standard, the following abbreviations apply.
2.2.1 DEC: Diarrheagenic Escherichia coli
2.2.2 EPEC: Enteropathogenic Escherichia coli
2.2.3 EIEC: Enteroinvasive Escherichia coli
2.2.4 ETEC: Enterotoxigenic Escherichia coli
2.2.5 STEC: Shiga toxin-producing Escherichia coli
2.2.6 EHEC: Enterohemorrhagic Escherichia coli
2.2.7 EAEC: Enteroaggregative Escherichia coli
2.2.8 escV: gene encoding LEE-encoded type Ⅲ secretion system factor
2.2.9 eae: gene encoding intimin for Escherichia coli attaching and effacing
2.2.10 bfpB: bundle-forming pilus B;
2.2.11 stx1: Shiga toxin one
2.2.12 stx2: Shiga toxin two
2.2.13 lt: heat-labile enterotoxin
2.2.14 st: heat-stable enterotoxin
2.2.15 stp (stIa): heat-stable enterotoxins initially discovered in the isolates from pigs
2.2.16 sth (stIb): heat-stable enterotoxins initially discovered in the isolates from human
2.2.17 invE: invasive plasmid regulator
2.2.18 ipaH: invasive plasmid antigen H-gene
2.2.19 aggR: aggregative adhesive fimbriae regulator
2.2.20 uidA: β-glucuronidase gene
2.2.21 astA: enteroaggregative heat-stable enterotoxin A
2.2.22 pic: protein involved in intestinal colonization
2.2.23 LEE: Locus of enterocyte effacement
2.2.24 EAF: EPEC adhesive factor
3 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
3.1 Constant temperature incubator: 36℃±1℃,42℃±1℃.
3.2 Refrigerator: 2℃~5℃.
3.3 Thermostatic water bath: 50℃±1℃, 100℃ or adapting 1.5mL or 2.0mL metal bath (95℃~100℃).
3.4 Electronic balance: with sensibility of 0.1g and 0.01g.
3.5 Microscope: 10×~100×.
3.6 Homogenizer.
3.7 Oscillator.
3.8 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipettor and pipette tip.
3.9 Aseptic homogenizing cup or aseptic homogenizing bag: with capacity of 500mL.
3.10 Aseptic culture dish: 90mm in diameter.
3.11 pH meter or precision pH paper.
3.12 Microcentrifuge tube: 1.5mL or 2.0mL.
3.13 Inoculating loop: 1μL.
3.14 Low-temperature high-speed centrifuge: rotation speed ≥13000 r/min, temperature control at 4℃~8℃.
3.15 Microbial identification system.
3.16 PCR instrument.
3.17 Micropipettor and pipette tip:0.5μL~2μL, 2μL~20μL, 20μL~200μL, 200μL~1000μL.
3.18 Horizontal electrophoresis apparatus: including power supply, electrophoresis tank, glue-manufacturing tank (with length >10cm) and comb.
3.19 8-tube strip and 8–cover strip (flat cover/convex cover)
3.20 Gel-imaging apparatus.
4 Media and Reagents
4.1 Nutrient broth: see A.1.
4.2 Enterobacteria enrichment broth: see A.2.
4.3 Maconkey agar (MAC): see A.3.
4.4 Eosin methylene blue agar (EMB):see A.4.
4.5 Triple sugar iron (TSI)agar: see A.5.
4.6 Peptone water and indole reagent: see A.6.
4.7 Semi-solid agar: see A.7.
4.8 Urea agar (pH 7.2): see A.8.
4.9 Potassium cyanide(KCN) medium: see A.9.
4.10 Oxidase reagent: see A.10.
4.11 Gram stain solution: see A.11.
4.12 BHI broth: see A.12.
4.13 Formalin (including 38%~40% of formaldehyde).
4.14 Identification kit.
4.15 Escherichia coli diagnostic serum.
4.16 Aseptic deionized water.
4.17 0.85% aseptic normal saline.
4.18 TE(pH8.0): see A.13.
4.19 10×PCR reaction buffer: see A.14.
4.20 25mmol/L MgCl2.
4.21 dNTPs: dATP, dTTP, dGTP and dCTP with concentration of 2.5mmol/L.
4.22 Taq enzyme in 5U/L.
4.23 Primer.
4.24 50×TAE electrophoretic buffer: see A.15.
4.25 Agarose.
4.26 Ethidium bromide(EB) or other nucleic acid dye.
4.27 6×loading buffer solution: see A.16.
4.28 Marker: Molecular weight includes bands of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp and 1500bp.
4.29 Diarrheagenic Escherichia coli PCR kit.
5 Examination Procedures
See Figure 1 for examination procedures of diarrheagenic Escherichia coli.
Figure 1 Examination Procedures of Diarrheagenic Escherichia Coli
6 Operation Steps
6.1 Sample preparation
6.1.1 Solid or semi-solid sample
Foe solid or semi-solid sample, weigh 25g of examined sample by aseptic operation, add it into a homogenizing cup with 225mL of nutrient broth to homogenize by a spinning blade-type homogenizer at 8000r/min~10000r/min for 1min~2min; or add the examined sample into a homogenizing bag with 225mL of nutrient broth to homogenize by a slap-type homogenizer for 1min~2min.
6.1.2 Liquid sample
Weight 25mL of examined sample by aseptic technique, add it into an aseptic conical flask with 225mL of nutrient broth (proper amount of aseptic glass beads may be preset in the flask), and then oscillate the flask to mix them well.
6.2 Enrichment
Culture the homogeneous sample solution prepared in 6.1 at 36℃±1℃ for 6h. Take 10μL of the solution, inoculate it into 30mL enterobacteria enrichment broth tube, and then culture at 42℃±1℃ for 18h.
6.3 Isolation
Carry out streak inoculation for the enrichment broth into MAC and EMB agar plates, culture them at 36℃±1℃ for 18h~24h, and then observe colony characteristics. On the MAC agar plate, the typical colony decomposing lactose is brick red to peach while the colony not decomposing lactose is colorless or light pink. On the EMB agar plate, the typical colony decomposing lactose is purple black with or without metallic luster in center while the colony not decomposing lactose is colorless or light pink.
Contents of GB 4789.6-2016
Foreword I
1 Scope
2 Terms, Definitions and Abbreviations
3 Apparatus and Materials
4 Media and Reagents
5 Examination Produces
6 Operation Steps
7 Result Report
Appendix A Media and Reagents
