A selective chromogenic plate, YECA, for the detection of pathogenic Yersinia enterocolitica: specificity, sensibility and capacity to detect pathogenic Y. enterocolitica from pig tonsils

A new selective chromogenic plate, YECA, was tested for its specificity, sensitivity and accuracy to detect pathogenic Y. enterocolitica from pig tonsils. We tested a panel of 26 bacterial strains on YECA and compared it to PCA, CIN and YeCM media. Detection of pathogenic Y. enterocolitica was carried out on 50 pig tonsils collected in one slaughterhouse. Enrichment was done in PSB and ITC broths. Streaking on YECA and CIN was done in direct, after 24H incubation of ITC, after 48H incubation of PSB and ITC. All the plates were incubated at 30°C during 24 hours. Presence of typical colonies on CIN and YECA was checked and isolates were biotyped. Pathogenic Y. enterocolitica strains showed an important growth on YECA with small and red fuchsia colonies while biotype 1A exhibited very few violet colonies. Enrichment in ITC during 48H gave the best performance for detecting positive samples in pathogenic Y. enterocolitica and YECA could detect directly pathogenic Y. enterocolitica strains (2, 3 and 4). Combination of ITC enrichment and YECA detection generates a time-saver by giving a positive test for pathogenic Y. enterocolitica in 72 hours. Introduction In 2009, yersiniosis was, for the sixth consecutive year, the third most frequently reported human zoonosis in the Europe, with a total of 8,354 confirmed cases (EFSA, 2010). In France and most other countries worldwide, biotype 4 is the most prevalent biotype isolated from humans (69%), followed by biotype 2 (30%) and biotype 3 (Savin and Carniel, 2008). Pigs are considered the principal reservoir for the types of Y. enterocolitica pathogenic to humans. Pigs do not develop clinical signs, but they do carry Y. enterocolitica in their oral cavity, on tongues and tonsils, and in lymph nodes, and excrete this bacterium in their feces (Thibodeau et al., 1999). Detection of Yersinia is carried out by using ISO 102732003 method. This method is recommended for both food and pig tonsil analyses (EFSA, 2007) but involves time-consuming enrichment steps in two broths, PSB and ITC, followed by plating on 2 selective media, SSDC and CIN (De Boer, 1992). Biochemical tests on isolates are also necessary to confirm Yersinia and to determine the biotypes. In this work, we tested a new selective chromogenic plate, Yersinia enterocolitica agar (YECA), for its specificity, sensitivity, and accuracy to detect pathogenic Y. enterocolitica from pig tonsils. Material and Methods Specificity of YECA. The specificity of YECA (AES Chemunex, Combourg, France) was tested against 26 strains listed in table 1. Each culture was streaked on CIN agar plate, on YeCM medium (Weagant, 2008) and, on YECA plates. If growth of bacteria was observed on plate, importance of growth in a scale from 1 to 5 and characteristics of the colonies were noted. Sensitivity of YECA. Y. enterocolitica strains from biotype 1A, 2, 3, and 4 were incubated in 5ml of BHI broth during 24h at 30°C. A 10-fold serial dilution of the cultures was done and 100μl of each dilution were spread on YECA and compared with PCA, CIN and YeCM media. Enumeration of the colonies was then performed after incubation of the plates at 30°C for 24hours. Detection of pathogenic Yersinia enterocolitica from pig tonsils. 50 pig tonsils were collected from a slaughterhouse. From each tonsil, 10 g were cut in small pieces and put into a bag containing 90 ml of PSB broth. After stomaching, 10μl were streaked directly onto YECA and CIN plates; and 1 ml was transferred in 9 ml of ITC broth. PSB and ITC were incubated at 25°C for 48 hours, before a second streaking onto YECA and CIN. In addition, after 24 hours of enrichment in ITC broth, an extra streaking on YECA and CIN was performed. All the plates were incubated at 30°C for 24 hours. Presence of typical colonies on CIN and on YECA was checked. At least two typical colonies per plate were streaked on