Macconkey agar

The 25 samples that were collected had been isolated from different lesion sites (Table-1). The isolates were seeded on 8% sheep blood agar (Sigma-Aldrich, Darmstadt, Germany) and MacConkey agar (Neogen Corporation, São Paulo, Brazil) at 37°C under aerobic conditions and characterized morphologically and biochemically as described by Quinn et al. [14 ].

E. coli was isolated and identified according to Lee and Nolan [11 ]. Briefly, all the collected samples were pre-enriched in buffered peptone water (Oxoid, UK) and incubated aerobically at 37°C for 24 h. Then, a loopful of the broth culture was inoculated onto MacConkey agar (Neogen, US) and eosin methylene blue agar (LabM, UK) plates and reincubated at 37°C for 24 h. The isolated colonies were identified morphologically and biochemically (oxidase strips and triple sugar iron agar from Oxoid; urea, Simmon citrate agar, and peptone water from LabM; and Kovac reagent from HiMedia, India).

From each sample, fecal material was streaked onto MacConkey agar (Neogen, Michigan, USA) using a sterile cotton-tipped swab and incubated overnight at 37 °C. Five lactose-fermenting (bright pink) colonies with typical E. coli morphology were randomly selected from the MacConkey agar plate. These colonies were subcultured on horse blood agar plates (Neogen, Michigan, USA), incubated overnight at 37 °C, and tested for production of tryptophanase (indole) using the spot indole test (Miller and Wright 1982 (link)). Lactose- and indole-positive isolates with typical colony morphology (bright pink on MacConkey agar, blue in spot indole test and single-colony types) were considered E. coli. Confirmed isolates of E. coli were transferred to 2-mL microtubes (SARSTEDT, Nümbrecht, Germany) containing 0.5 mL serum broth supplemented with 15% glycerol. The microtubes were placed in a freezer at − 80 °C. One of the five frozen isolates of E. coli from each calf sample was selected at random and sent frozen (on dry ice) to the National Veterinary Institute, Uppsala, Sweden. Directly upon arrival, isolates were transferred to a freezer at − 20 °C and stored until further testing.

Escherichia coli strains were isolated in the Basic and Applied Bacteriology Laboratory at Londrina State University (Biosafety level 2) from 98 commercial refrigerated chicken carcass (35 chicken carcasses from PR, 23 chicken carcasses from SC, and 40 chicken carcasses from RS), sold in southern Brazil from 2013 to 2014. Each chicken carcass was rinsed into the sterile packaging with 100 mL of Brain Heart Infusion (Himedia Laboratories Pvt. Ltd., Mumbai, India). After homogenization, 0.1 mL of the mixture was smeared onto MacConkey agar (Neogen Corporation Lansing, Michigan) and Violet Red Bile Lactose agar (Oxoid Ltd., Basingstoke, Hants, UK) by the pour plate method. Colonies suspected to be E. coli were confirmed by biochemical testing using EPM-MILi and Simmons Citrate agar (PROBAC, Brazil). After biochemical confirmation, one to five strains were collected from each chicken carcass and subsequently analyzed for the genotypic characteristics of ExPEC virulence factors and phenotypic resistance. Only strains that showed difference in those characteristics were selected for further analysis.

Escherichia coli was isolated and identified according to Nolan et al. [1 ]. Briefly, all the collected samples were pre-enriched in buffered peptone water (Lab M, UK) and incubated aerobically at 37°C for 24 h. A loopful of the broth culture was inoculated onto MacConkey agar (Neogen, US) and eosin methylene blue agar (Lab M) plates, which were incubated at 37°C for 24 h. The isolated colonies were identified morphologically and biochemically (oxidase strips and triple sugar iron agar were from Oxoid, UK; urea, Simmons’ citrate agar, and peptone water were from Lab M; and Kovacs reagent was from HiMedia, India) [1 ]. In addition, antisera against somatic (O) antigens (Denka Seiken Co., Tokyo, Japan) were used for serotyping isolated E. coli following the manufacturer’s instructions.