Api 20e biochemical test strips

All stx-positive isolates were confirmed biochemically as E. coli using API® 20E biochemical test strips (bioMérieux, Lyon, France). The O serogroup of each isolate was preliminary screened using a PCR method with O antigen specific primers [48 (link)] and confirmed by using all O1-O188 E. coli antisera (SSI Diagnostica, Hillerød, Denmark). The entire coding sequence of the fliC gene was amplified by PCR, and then sequenced to determine the H type, as described previously [19 (link)].

Fecal samples from diarrheal patients, healthy carriers, animals, and meats were collected from April 2009 to March 2019 in different regions of China. STEC strains were isolated and confirmed by the methods as previously described [49 (link)]. Briefly, the samples were enriched in EC broth and then tested for the presence of stx₁/stx₂ genes by PCR. stx-positive samples were inoculated into two selective media, i.e., CHROMagar^TM ECC agar and CHROMagar^TM STEC agar (CHROMagar, Paris, France), for isolation of STEC strains, as described previously [42 (link)]. After overnight incubation at 37 ℃, presumptive colonies were picked and tested for stx genes by single a colony duplex PCR assay. API 20E biochemical test strips (bioMérieux, Lyon, France) were used for a confirmatory test. The stx₁/stx₂ subtyping was initially conducted by amplifying, sequencing the complete stx₁/stx₂ genes, and comparing against known stx subtypes, as described previously [5 (link)]. STEC strains carrying the stx_2e subtype were selected for subsequent study.

The method of McEvoy et al. (2003) was used for isolation of E. coli O157:H7. Twenty‐five gram of kimchi was transferred to a sterile stomacher bag containing 225 ml of modified E. coli broth with novobiocin (mEC Broth; Merck), homogenized for 2 min with a stomacher and enriched by incubating at 37°C for 24 hr. One loop of the enrichment broth was streaked onto a plate of Sorbitol MacConkey Agar with cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L) (CT‐SMAC, Oxoid) and incubated at 37°C for 24 hr. For a second isolation, colonies formed on the CT‐SMAC agar plate were streaked onto eosin methylene blue agar plates (EMB, Becton, Dickinson Co.) and phenol red agar with 4‐methylumbelliferyl‐β‐D‐glucuronide (PRS‐MUG) and incubated at 37°C for 24 hr. Presumptive positive isolates on EMB and PRS‐MUG agar were tested for O157 and H7 antigens using the latex agglutination test (Remel^™ Wellcox^™E. coli O157:H7). Latex agglutination‐positive isolates were subjected to the following tests: H₂S production, indole production, Voges–Proskauer test, citrate utilization and lysine decarboxylase and ornithine decarboxylase test by using API 20E biochemical test strips (bioMérieux, La Balme les Grottes, France), cellobiose fermentation by API 50CHL medium (bioMérieux, La Balme les Grottes, France), and carbohydrate fermentation in triple sugar iron agar slants (TSI, Becton, Dickinson Co.).