Api coryne test

Thirty-seven T. pyogenes strains isolated from various animal hosts and different suppurative infections were used in this study (12 from goats, 8 from cattle, 8 from pigs, 2 from sheep, and 7 from European bison). All strains were selected from the strain collection of the Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences (an origin and a source of strains are shown in Figure 3 and Table S2 of Supplementary Materials). The studied strains were isolated from individual animals from different farms, with the exception of nine strains originating from three goats (strains 28/K, 30/K, 31/K, 34/K, strains 26/K, 27/K and strains 21/K, 22/K, 54/K were isolated from the goat 3, the goat 2 and the goat 1, respectively). All strains were identified using the API Coryne test (BioMérieux, Marcy l’Etoile, France) and also by the species-specific plo gene PCR, as described previously [23 (link),24 (link)].
Bacteria were cultured on Columbia Agar supplemented with 5% sheep blood (Graso Biotech, Starogard Gdański, Poland) at 37 °C under microaerophilic conditions for 48 h. Then, a pure culture of each strain was prepared on Tryptic Soy Broth (TSB; BioMérieux, Marcy l’Etoile, France), and after 24 h incubation, it was used for a template DNA extraction.

We cultured collected swab specimens on tellurite-containing agar medium in a 35°C incubator for 24‒48 hours (3 ). If black colonies grew, we initially tested them by Gram stain to identify gram-positive bacilli (3 ). We used the API Coryne Test (bioMérieux, https://www.biomerieux.com) to identify species and biovars for each subculture (3 ). We tested subcultures for expression of the diphtheria toxin by using the modified Elek test (24 (link)).
We conducted quadruplex, real-time, reverse transcription PCR (qRT-PCR) directly on throat swab specimens and aliquots of skim milk, tryptone, glucose, and glycerin medium to identify C.diphtheriae, C.ulcerans, or C. pseudotuberculosis and the diphtheria toxin gene according to published methods (3 ,25 (link)). DNA was extracted by using the QIAmp DNA Extraction Kit (QIAGEN, https://www.qiagen.com) (26 (link)). Primers and probes targeted 2 rpoB genes, the tox gene, and the green fluorescent protein gene (gfp), which we used as internal positive controls.

The isolates were biotyped using the API Coryne test (BioMerieux, France) according to the manufacturer’s instruction. Toxigenicity was determined by conventional and modified Elek tests according to the WHO manual [12 ]. The presence of the diphtheria toxin gene was investigated by PCR as described previously [13 (link)].