Emb agar

We screened Escherichia coli and Klebsiella pneumoniae as target nosocomial pathogens from hospital wastewater samples. Fifty milliliters of hospital wastewater from sample bottle were transferred into conical centrifuge tube. Then, the samples were concentrated by centrifugation with 3000 rpm speed for 10 min to generate intact bacterial pellets.
A loop of bacterial pellets was inoculated to Eosin Methylene Blue (EMB) agar (Oxoid, Basingstoke, UK) and MacConkey agar (Oxoid, Basingstoke, UK), at 37 °C overnight to get cultural characteristics of Escherichia coli and Klebsiella pneumoniae colonies, respectively. Colonies showing metallic sheen in EMB agar and mucoid lactose fermenter colonies in MacConkey agar were suspected. In case the colonies were not isolated, the subculture was carried out for obtaining pure culture.
Vitek2 system was used to identify Escherichia coli and Klebsiella pneumoniae and performed antibiotic susceptibility testing (bioMérieux, Marcy l’Etoile, France). Beta-lactam antibiotics tested included ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, cefazolin, ceftazidime, ceftriaxone, cefepime, and meropenem (Table S1). Antibiotic susceptibility test results were interpreted by the Clinical Laboratory Standard Institute 2022 guideline.

In total, 116 APEC isolates recovered from 400 different samples were assayed. Samples were obtained from liver, lungs, air sacs and spleen from chicken with typical lesions of colisepticemia and were collected from 28 different broiler farms located in different geographic areas of Dakahlia Governorate in 2014 and 2015. Samples were cultured onto eosin methylene blue (EMB) agar (Oxoid, Basingstoke, UK) and incubated for 18–24 h at 37 °C. Typical colonies were subcultured onto MacConkey agar (Oxoid, Basingstoke, UK) and were biochemically confirmed using API 20E system (BioMérieux, Marcy-l’Étoile, France). Once identified, the isolates were preserved at −70 °C in brain heart infusion broth containing 20% glycerol (vol/vol) for further studies. Serotyping of E. coli isolates was performed using rapid diagnostic E. coli antisera sets (Denka Seiken Co., Japan) using 8 polyvalent, 43 monovalent somatic, and 22 flagellar antisera. Bacterial DNA for PCR analysis was prepared by boiling a bacterial culture in 300 µl of distilled water for 10 min, followed by centrifugation for 5 min at 10,000×g. A volume of 1 µl of the supernatant was used as a template for each 25 µl PCR mixture.

For model development, the popularity of yogurt samples and results of physicochemical (high pH value) and microbiological analyses of yogurt were considered. Drinking and regular yogurt were purchased from an offline market (Seoul, Korea) and aseptically divided into 30 mL and 10 g, respectively, into 50 mL conical tubes (SPL Life Science Co., Daejeon, Seoul). LM and the cocktail of E. coli strains were independently inoculated into drinking (4~5 log CFU/g) and regular yogurts (5~6 log CFU/g). Each sample was then stored at 4, 10, 17, 25, and 36 °C until no colonies were detected for up to 21 days. At a specific time, each yogurt sample was homogenized with sterilized 0.1% peptone water for 120 s using a stomacher. Then 1 mL of the aliquot of the homogenate was serially diluted ten-fold with 0.1% peptone water and spread onto PALCAM agar (Oxoid, Basingstoke, Hampshire, UK) for LM and EMB agar (Oxoid, Basingstoke, Hampshire, UK) for EHEC, which were incubated at 36 ± 1 °C for 24 h to analyze the change in pathogen populations.

A loopful from each organ was cultured on MacConkey’s agar (Oxoid, UK) plates incubated aerobically at 37°C for 24 h. Suspected pink colonies were picked up, streaked, and incubated overnight at 37°C on eosin methylene blue (EMB) agar (Oxoid). Biochemical tests, such as catalase, oxidase, urease, methyl red, Voges–Proskauer, citrate utilization, indole, and triple sugar iron agar, were used to identify suspected E. coli colonies [18 ]. Slide agglutination with polyvalent and monovalent fast diagnostic sets of E. coli antisera (Denka Seiken Co., Ltd., Japan) was used to identify serotypes of E. coli isolates [19 ].

A total of 320 stool samples were included in the study. One stool sample was collected from each patient in sterile container and transported to the microbiology laboratory in cool box for immediate examination. The samples were examined for consistency, presence of blood, mucous or occult blood. The samples were inoculated into MacConkey broth for enrichment at 37 °C for 24 h. The enrichments were streaked onto MacConkey agar (Oxoid, Basingstoke, United Kingdom). and incubated for 24 h at 37 °C. After that, lactose fermenters isolates were subcultured onto eosin methylene blue agar (EMB) agar (Oxoid, Basingstoke, United Kingdom). Colonies producing greenish metallic sheen on EMB agar were examined by biochemical tests including; IMViC (indole, methyl red, Voges-Proskauer, citrate utilization), urease agar, sugar fermentation and motility tests. In addition the identified E. coli isolates were confirmed by chromogenic media (CHROMagar™ Orientation, Paris, France). Samples containing E. coli only were included. Hemolytic activity was tested by culturing of isolates on blood agar. E. coli isolates were kept in trypticase soy broth (Oxoid, UK) with sterilized 20% glycerol at − 20 °C for further investigations.