GB 4789.6-2016National food safety standard -- Microbiological examination of food -- Examination of diarrheagenic Escherichia coli (English PDF)

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

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°C±1°C for 6h. Take

10µL of the solution, inoculate it into 30mL enterobacteria enrichment broth tube, and then culture at 42°C±1°C for 18h.

6.3 Isolation

Carry out streak inoculation for the enrichment broth into MAC and EMB agar plates, culture them at 36°C±1°C 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.

6.4 Biochemical test

6.4.1 Select 10~20 suspicious colonies from the plates (select all colonies in case of

10 below), therein lactose-fermented, lactose non-fermented and delayed fermented colonies shall be picked up to inoculate TSI slants respectively. Simultaneously, inoculate these cultures to peptone water, urea agar (pH 7.2) and KCN broth respectively. Culture them at 36°C±1°C for 18h~24h.

6.4.2 In the case that TSI slant produces acid or fails to produce acid, bottom layer produces acid, indole is positive, as well as both H2S and urase are negative, the culture is Escherichia coli. In the case that TSI slant and bottom layer fails to produce acid, or any one of H2S, KCN and urea is positive, the culture is non-Escherichia coli.

If necessary, gram stain and oxidase test shall be carried out. The Escherichia coli is gram negative bacillus and oxidase is negative.

6.4.3 If biochemical identification kit or microbial identification system is adopted, the depurated colonies shall be picked from nutrient agar plate to prepare bacterial suspension with appropriate turbidity by aseptic diluent, and then identified by biochemical identification kit or microbial identification system.

6.5 PCR confirmatory test

6.5.1 Adopt the colonies with biochemical reaction in conformity to Escherichia

coli characteristic to carry out PCR confirmatory test.

Note: PCR laboratory location planning, basic working principles and attentions shall refer to the "Construction Standard of Center for Disease Control and Prevention" (JIANBIAO 127-2009) and the Appendix (Working Guideline of Clinical Gene

Amplification Examination Laboratory in Medical Institution) of "Management

Method for Clinical Gene Amplification in Medical Institution" issued by the

National Health and Family Planning Commission of the People's Republic of China (the original Ministry of Health of the People's Republic of China) (2010).

6.5.2 Apply 1µL inoculating loop to scrape the colonies cultured for 18h~24h from

nutrient agar plate or slant, suspend them in 200µL of 0.85% aseptic normal saline, sufficiently scatter and prepare bacterial suspension, centrifuge at 13000r/min for

3min, and then discard the supernatant. Add 1mL of aseptic deionized water to sufficiently mix the thallus well, and maintain in 100°C water bath or metal bath for

10min. After cooling by ice bath, centrifuge at 13000r/min for 3min, collect supernatant and dilute it by aseptic deionized water in the proportion of 1: 10, and then adopt 2µL of which to serve as the template for PCR detection. All the treated

DNA templates are directly used for PCR reaction or stored temporarily at 4°C and subjected to PCR reaction; otherwise, they shall be preserved at -20°C below for standby (within one week). Bacterial genomic extraction kit may also be adopted for extracting bacterial DNA, which shall be operated according to the instruction of bacterial genomic extraction kit.

6.5.3 For each PCR reaction, EPEC, EIEC, ETEC, STEC/EHEC and EAEC

standard strains shall be adopted as positive control. Meanwhile, Escherichia coli

ATCC 25922 or equivalent standard bacterial strain shall be adopted as negative control, and aseptic deionized water as blank control, controlling PCR system pollution. See Table 1 for characteristic genes of diarrheagenic Escherichia coli.

sufficiently mix them well, slightly pour the mould with comb, the length of gelation shall be greater than 10cm and the thickness should be 3mm~5mm. Inspect whether bubble exists below or among comb, if any, exhaust the bubble from agarose gel with disposable tip. After the agarose gel entirely coagulates and hardens, slightly pull out the comb, carefully put glue block and glue bed inside the electrophoresis tank, and place the sample hole at positive electrode. Add 1×TAE electrophoretic buffer into the electrophoresis tank, with liquid level 1mm~2mm above the glue surface. Well- mix the 5µL of PCR product with 1µL of 6×loading buffer solution, suck the mixed solution with micro pipettor and vertically stretch into the glue hole below liquid level and carefully add the sample into the hole; Add the PCR reaction product of positive control into the last swimming lane; add 2µL of molecular weight Marker into the first swimming lane. Switch on the electrophoresis apparatus, calculate and set the voltage value according to the Formula: voltage = the distance between the positive and negative electrodes of electrophoresis tank (cm)×5V/cm; turn on the switch of voltage, and the electrophoresis starts when bubbles appear in platinum at positive and negative electrodes. After electrophoresis for 30min~45min, cut off the power.

Take out the gel and put it into gel-imaging apparatus to observe the result and photograph and record the date.

6.5.7 Result judgment. The blank control in electrophoresis result shall be free from band, negative control only appears uidA band amplification, and positive control appears all target bands, in this case, the PCR test result shall be true. According to the size of target bands in electrophoretogram, judge the kind of target band, record the kind of target band in each swimming lane, and search the category of diarrheagenic Escherichia coli corresponding to different target band kind and combination in Table 5.

6.5.8 If commercialized PCR kit or multiple PCR(MPCR) kit is adopted, the operation and result judgment shall be conducted according to its instruction.

6.6 Serological test(optional)

6.6.1 Adopt the strain confirmed as diarrheagenic Escherichia coli via PCR test for

serological test.

Note: the O antigen and H antigen shall be identified according to the operating instructions provided by the manufacturer. Where the operating instructions provided by the manufacturer may be deviated from the descriptions below, the former one shall prevail.

6.6.2 O antigen identification

6.6.2.1 Presumptive test: pick the colony confirmed as diarrheagenic Escherichia

coli via biochemical test and PCR test on nutrient agar plate; according to the category of diarrheagenic Escherichia coli, adopt the univalent or multivalent OK serum of Escherichia coli to carry out slide agglutination test. Where agglutinated with a certain kind of multivalent OK serum, conduct agglutination test for the univalent OK serum involved in the multivalent serum. See Table 6 for the O antigen group included in Diarrheagenic Escherichia coli. If agglutination reaction presents for certain univalent OK serum, the presumptive test is positive.

6.6.2.2 Confirmatory test: prepare O antigen suspension with 0.85% aseptic normal

saline, and dilute it to the concentration equivalent to MacFarland No. 3 turbidimetric tube. Dilute the O-serum with original valence of 1: 160~1: 320 by 0.5% of saline water to 1: 40. Mix the diluted serum with antigen suspension in 10mm×75mm test tube equivalently and carry out single-tube agglutination test. After mixing well, place it in a water bath at 50°C±1°C for 16h to observe the result. If agglutination appears, it may be confirmed as O antigen.

6.6.3 Identification of H antigen

6.6.3.1 Adopt strain and inoculate into semi-solid agar tube by stabbing, culture it at

36°C±1°C for 18h~24h, adopt one loop of top culture to inoculate into BHI liquid medium, and culture it at 36°C±1°C for 18h~24h. Add formalin into it until the concentration reaches 0.5% to carry out slide agglutination or tube agglutination test.

6.6.3.2 If there is no obvious agglutination in both to-be-tested antigen and serum,

......