Api listeria test

The biochemical confirmation reactions were performed with the API Listeria test (bioMérieux, France) according to the instructions of the manufacturer using bacterial cultures cultivated freshly on sheep blood agar plates (OXOID, UK). Reading of the API test strips was evaluated with the mini-API instrument (bioMérieux, France).

The soil, silage, fecal, and manure composite samples were weighed, diluted with 1% buffered peptone water (BPW), and pummeled (BagMixer; Weymouth, MA, Interscience Laboratories, Inc.) for bacterial analysis as formerly pronounced by Latorre et al. (2010 (link)). All samples were pre-enriched by adding 5 ml of BPW or 5 ml of milk to 5 ml of half Fraser broth (Oxoid, Basingstoke, UK) and incubated for 24 h at 37 °C. For the enrichment, 1 ml of the pre-enriched half Fraser broth was added to 9 ml of Fraser broth (Oxoid, Basingstoke, UK) for each sample and incubated at 37 °C for 24 h. Each enriched Fraser broth culture was cultured onto Palcam agar (Oxoid, Basingstoke, UK) and incubated for 48 h at 37 °C. Presumptive L. monocytogenes colonies (gray with black center) were biochemically identified using the following tests: catalase test, oxidase test, evaluation of hemolysis type, motility at 25 °C and 37 °C, in addition to sugar fermentation test (Van Kessel et al. 2004 (link)). The strains expressing these standard features were further tested using the API Listeria test (BioMerieux) for confirmation.

Detection of L. monocytogenes was performed as described by ISO 11290-1 for the isolation of such bacteria from food [20 (link)]. First for the enrichment of L. monocytogenes, 25 g of each poultry sample was diluted in 225 ml of Half Fraser broth (Oxoid, UK) and homogenized in a blender for 2 minutes. The hand swab samples were transferred to 10 ml of Half Fraser broth. Homogenates of poultry samples and swabs were incubated at 30 °C for 24 h. After that, 0.1 ml of pre-enriched culture was added to 10 ml of Fraser broth and incubated at 30 °C for 24 h. Each Fraser broth culture was streaked onto Palcam agar (Oxoid) and incubated at 37 °C for 48 h. Approximately five colonies of the growing Listeria specieswere purified and underwent further biochemical identification using catalase test, oxidase test, sugar fermentation test, and evaluation of hemolysis type [21 (link)]. The biochemically confirmed strains of L. monocytogenes in the present study were further verified using the API Listeria test (BioMerieux).