Api 20e test system

Sixty-four frozen tissue samples from 50 animals (intestines (with fecal content) (n = 46), liver (n = 6), lung (n = 9), and kidney (n = 3)) were used to attempt cultivation of E. coli. To compensate for sub-lethal injuries of the bacteria due to the freezing process of organs at -80°C, all samples were initially incubated over night at 37°C in brain heart infusion (BHI) broth (Oxoid, Germany). BHI broth was streaked on Columbia blood agar (5% blood), Gassner agar and Chrom orientation agar (Oxoid, Germany) and incubated over night at 37°C. Purple colonies from Chrom orientation agar were confirmed as E. coli by conventional biochemical tests as described previously [24 (link)]. Ability of haemolysis of corresponding colonies was assessed on blood agar. Where biochemical tests revealed ambiguous results, bacterial species were identified with the Api 20E test system (Biomérieux, Germany). One E. coli isolate per sample was picked and used for further analyses, except in cases E. coli colonies showed two various morphologies on Gassner agar. Then two E. coli isolates per sample were taken. All isolates were stored at -80°C in BHI broth with 10% glycerol until further use.

The strains were tested for distinctive enzymatic reactions and metabolic routes utilizing API 20 E test system (bioMerieux, France), according to the manufacturer’s instructions.

The laboratory procedures for culturing Yersinia bacteria were conducted at the Richard Lugar Center for Public Health Research, Tbilisi, Georgia, following the standard operating procedures approved by NCDC’s QMS committee (SOP-GBG -116- Identification of Yersinia species _V3.0, 2020). Suspensions of small intestine samples were inoculated into 1% peptone water followed by growth for 28 days at 4 °C. The inoculum was plated every 5th day on Endo and CIN agar (bioMérieux, Marcy-l’Étoile, France), and plates were incubated at 28 °C for 7 days. Bacterial colonies with a morphology resembling Yersinia (round shape, 0.5–1 mm diameter size, and grayish-pinkish color; a “bulls-eye” appearance on CIN agar) were sub-cultured on Endo agar plates at 28 °C for 24–48 h. The colonies obtained were analyzed microscopically after Gram staining. BD Phoenix (Becton & Dickinson, Eysins, Switzerland) and API (Analytical Profile Index, bioMérieux, France) tests were used according to the manufacturers’ guidance for further analysis. Identification of Y. enterocolitica was conducted with the use of the API20E test system (Analytical Profile Index, bioMérieux, France) and the BD Phenix50 Automatic System using a Gram-Negative ID Panel (Becton & Dickinson, Switzerland) according to the manufacturers’ guidance for further analysis.

All samples of fresh droppings were screened for Salmonella by the alternative technique VIDAS Easy Salmonella (bioMérieux SA, Lyon, France). This technique is based on the detection of specific Salmonella proteins by immune-fluorescence after primary and secondary enrichment followed by lysis of the bacteria by heating at 100 °C. Primary enrichment consists of incubating, at 37 °C for 22 h, a mixture of 25 g of fresh droppings samples and 225 mL of Buffered Peptone Water (Biokar Diagnostics, Beauvais, France). After incubation, 100 μL of the mixture was transferred into 10 mL of Salmonella Xpress 2 (SX2) broth (bioMérieux SA) and incubated at 41.5 °C for 24 h. One milliliter of the mixture was boiled for 15 min, then cooled to room temperature. To detect Salmonella target proteins, a volume of 0.5 mL of the already-cooled mixture was transferred to an SLM chip and deposited at VIDAS (bioMérieux SA). The unheated SX2 broth is used for the isolation of Salmonella, from positive samples, on selective agar (XLD and SS) (Biokar Diagnostics, Beauvais, France). Confirmation is performed by urease test followed by API 20E system test (bioMérieux SA) according to the reference technique ISO 6579, 2017 [18 ].

A total of 61 Salmonella strains were isolated from hospitalized patients with gastroenteritis at a university hospital in Great Tunisia between 2010 and 2020. The isolation of Salmonella from the selective enrichment SX2 broth (bioMérieux SA, Lyon, France) previously inoculated by a clinical specimen and incubated at 37 °C for 24 h, on selective agar (XLD and SS) (Biokar Diagnostics, Beauvais, France), was conducted following the reference technique indicated in REMIC, 2018 [36 ]. The identification and confirmation of Salmonella strains were performed via the urease test followed by the API 20E system test (bioMérieux SA, Lyon, France) according to the reference technique indicated in REMIC, 2018 [36 ].