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Turk J. Pharm. Sci. 5 (2) 8994, 2008 Original Article OCCURRENCE OF YERSINIA ENTEROCOLITA IN TURKISH WHITE CHEESE CONSUMED IN KARABÜK REGIONTURKEY Gülderen YENTÜR 1*, Nurcay BÜYÜKGÜÇLÜ KOCAMAN 2, Buket ER 1, Aysel BAYHAN ÖKTEM 1 1 Gazi University, Faculty of Pharmacy, Department of Food Analysis, Etiler Ankara, TURKEY 2 Refik Saydam National Hygiene Center, The Ministry of Health, SıhhiyeAnkara, TURKEY Abstract In this study, occurence of a s pathogen Yersinia enterocolitica in white cheese was studied. 100 white cheese samples were collected from Karabük of which 50 samples from open markets, 30 samples from A, B, C firms and 20 samples from supermarkets. Occurrence of Yersinia enterocolitica in the samples were analysed by using the ISO method which is a conventional methodology for the investigation of foodborne pathogens. It has been isolated and identified in 8 samples (8%) out of 100 white cheese samples which may be considered as potential health hazard for public. Key words: Yersinia enterocolitica, white cheese Karabük Yöresinde Tüketime Sunulan Beyaz Peynirlerde Yersinia enterocolitica Varhginın Araştinlması Bu gahsmada gıda kaynaklı bir patojen olan Yersinia enterocolitica 'mn beyaz peynirlerde varligi araştinlmistır. Araştırmada, Karabük piyasasından sağlanan 3 farkh firmaya ait (A, B, C) 30 adet beyaz peynir örneği, 20 adet paketlenmemiş beyaz peynir örneği ve pazarlardan sağlanan 50 adet beyaz peynir örneği olmak üzere toplam 100 adet beyaz peynir örneğinde gahsilmistır. Örneklerde Yersinia enterocolitica'mn varhginın analizi iqin, gıda kaynaklı patojen mikroorganizmalann araştinlmasında konvansiyonel bir me tot olan ISO kullamlmistır. Yersinia enterocolitica, 100 beyaz peynir örneğinin 8 inde (%8) izole edilmiş ve tammlanmistır. Bu örneklerin halk sağligi acisından potansiyel zarar oluşturabileceği dusunülmektedir. Anahtar Kelimeler. Yersinia enterocolitica, beyaz peynir *Correspondance: Phone: Gülderen YENTÜR, Nurcay BÜYÜKGÜÇLÜ KOCAMAN, Buket ER, Aysel BAYHAN ÖKTEM INTRODUCTION Yersinia enterocolitica is a bacterium indigenous to the gastrointestinal tract of warmblooded animals and is associated with human and animal diseases (1, 2). Diarrhea, gastroenteritis and mesenteric lymphadenitis are the most frequent clinical manifestations of human yersiniosis (3). Several studies indicated that yersiniosis in humans was caused by consumption of contaminated food (4). Y. enterocolitica has been isolated from a variety of foods, including meat, raw oysters poultry, raw milk, pasteurized milk and dairy products, salads and vegetables, fruits and from water (1, 5, 6, 7), however strains 0:3, 0:8, 0:5,27 and 0:9 are the most commonly implicated in infection (8, 9). Milk (raw or pasteurized) and milk products, particularly cheese and ice cream, have frequently been found to be contaminated with Y.enterocolitica (10). Y. enterocolitica can grow to large numbers at refrigeration temperatures, so milk contaminated with that organism could become a significant health risk for consumers. The presence of Yersinia spp. in traditional fresh cheese could be attributed to different factors such as the use of raw milk and the eventual contamination from human handlers, environment and water (2, 9, 10, 11, 12). Turkish white cheese is probably the most popular, and economically the most important variety of cheese in Turkey. In the traditional or artisanal manufacture of Turkish white cheese, milk may or may not be pasteurized and the curd is handled extensively by the cheese maker. Cheesemilk was applied to pasteurization at 7274 o C for 1520 sec and ripened at 1215 o C for 3060 days and than storaged at 5 o C (13, 14). Our aim was to identify and detect Yersinia enterocolitica in Turkish white cheese samples consumed in Karabük Region. EXPERIMENTAL Samples Collection In this study, 100 white cheese samples of which 50 from open markets, 30 packaged from A, B, C firms and 20 unpackaged samples from supermarkets were analysed. All white cheese samples were collected from Karabük region. All the samples were placed in sterile glass jars and were carried to the laboratory in a cold container and were used for analysis, immediately (15,16). Analyses of Samples Samples were analysed for Y. enterocolitica using one of the methods prescribed by the ISO (16). Y. enterocolitica 0:8 strain WA, Y. enterocolitica 0:3 strain E 61264, Y. enterocolitica 0:9 BL 87/46 were used as reference strains in this study. Y. enterocolitica strains were provided by Hacettepe University Department of Food Engineering. In isolation process, Peptone, Sorbitol and Bile Salts (PSB) Broth were used as enrichment and Yersinia Selective Agar Base (Oxoid CM 653) was used as a selective agar. For detection of Y. enterocolitica, a sample of 25 g was weighed into sterile glass jars homogenized with 225 ml Peptone, Sorbitol and Bile Salts (PSB) Broth and incubated at 2225 o C for 5 days. Presence of the microorganism was determined using CefsulodinIrgasan Novobiocine (CIN; Oxoid CM 653, Oxoid selective supplement SR 109 E) Agar, incubated at 90 Turk J. Pharm. Sci. 5 (2) 8994, o C for 24 h. Five colonies that are thought to be Yersinia spp. (on Yersinia selective agar, dark red colonies surrounded by a transparent border) were picked from the plating media. After incubation at 30 o C for 24 h in Nutrient Agar (Difco ), organisms were submitted to Gram staining and the following biochemical tests: Catalase and oxidase tests, glucose fermentation with or without gas production, urease production, H 2 S production, presence of tryptophan deamination, lysine decarboxylase, ornitin decarboxylase, sucrose fermentation, rhamnose fermentation, citrate, esculin hydrolysis and indole production, Vogesproskauer test, methyl red test, motility at 2226 o C and 37 o C. RESULTS AND DISCUSSION Y. enterocolitica was isolated from 8 (16%) of the 50 samples which were collected from open markets. Y. enterocolitica was isolated from neither 20 unpacked samples which were collected from supermarkets nor 30 samples which were collected from A, B, C firms. Data presented in Table 1 indicate an overall Y. enterocolitica prevalence of 8% in cheese samples. Table 1. Prevalence of Y. enterocolitica in the examined white cheese The Number The Number of Samples of Samples Isolated Y. enterocolitica White cheese samples White Cheese Samples From Open Market Samples of Known A, B, C Firms Unpacked Samples From Supermarkets and Groceries Total samples No colonies observed No colonies observed 8 Prevalence of Y.enterocolitica % 8 (16%) 0 % 0 % 8 (8%) In the present study, the number ranging from 1 to 100 were given to all cheese samples. The first 30 cheese samples belong to firms (A, B, C). The rest of cheese samples, belong to unpacked cheese, sold in supermarkets and open markets. Existence of Yersinia enterocolitica was observed in 36, 39, 43, 49, 59, 61, 67 and 70 numbered cheese samples which are collected from open markets and its biochemical tests were performed. Samples number 67 and 70 were urease negative and were regarded according to the information of the method employed. In the method used (ISO,1994) with the nonpathogenic strains the possibility of negative urease activity was emphasized. The results of the biochemical tests are shown in Table 2. According to the results, proportion of Y. enterocolitica in 50 samples, which were collected from open markets, was very high (16%). These results may depend on presence of unpasteurised milk, contamination that may occure in production, during storage or because of nonhygienic conditions (17,18). Y. enterocolitica was not detected in the white cheese of known firms and in the unpacked white cheese of supermarkets and groceries. This shows that all processes are operated in hygiene rules from production to consumption. The results show that people are taking risk with respect to their health by consuming the white cheese made by villagers and sold in open markets. Several authors have reported the detection of Y. spp, and particulary Y. enterocolitica in the cheese in Turkey. Among these, there is some data reporting lower isolation rates than those reported in the present study. For instance, Sağun (19), has investigated and has determined the 91 Gülderen YENTÜR, Nurcay BÜYÜKGÜÇLÜ KOCAMAN, Buket ER, Aysel BAYHAN ÖKTEM presence of 2.4% Y. enterocolitica in a total of 41 white cheese examined. Some researchers reported that even higher isolation rations were detected compared to the results of this study. For instance, Aytaç and Özbaş (20) have studied the presence of Y. enterocolitica in white cheese and they have determined 19 out of 66 (28.8%) samples positive of Y. enterocolitica. Morever, Yücel et. al (21) have also studied cheese samples and they have examined the samples for the presence of Yersinia spp. In 100 samples, 14 were determined to be contaminated with Yersinia spp. Y. enterocolitica is the most commonly isolated species with the percentage of Table 2. Results of biochemical test Biochemical Tests Reference strains and positive samples 0:3 0:8 0: (z) Catalase Oxidase Glucose Lactose Gas production H 2 S Urease Tryptophane deaminase Lysine decarboxylase Ornithine decarboxylase Sucrose Rhamnose Citrate Esculine Indole Vogesproskauer 37 oc Vogesproskauer 25 oc Methly Red Motility test 37 o C Motility test 2226 oc (z): weak reaction (z) (z) 70 The occurrence of Y. enterocolitica in cheese samples has been investigated in several countries. For instance, Brindani and Freschi (22) have reported Y. enterocolitica in one of the 24 cheese samples (4.2%) in Italy. Boer and Kuik (23) have investigated 50 cheese samples and they have isolated Y. enterocolitica in one sample (2%) in Netherlands. Brodsky (24) has mentioned that he has isolated Y. enterocolitica in one sample of the 112 cheese samples (0.9%) in Canada. These findings have lower rates in comparison with our study. There are also reports stating the lack of Yersinia spp. among the samples that were studied by Tornadijo et al (25) in Spain and Karplyuk et al (26) in Russia. As a result, foodborne outbreaks have been reported in recent years. This has attracted our attention in terms of both the economic and public health consequences of microbial 92 Turk J. Pharm. Sci. 5 (2) 8994, 2008 contamination of processed dairy products. The need for the sanitary procedures during production and avoiding the contamination after pasteurization is of great importance. ACKNOWLEDGEMENT The authors would like to acknowledge Prof. Dr. Aykut AYTAÇ from Hacettepe University Department of Food Engineering for providing the reference strains. REFERENCES 1. Falcao, D. P., Occurrence of Yersinia spp. in Foods in Brazil, International Journal of Microbiology, 14,179182, Hamama, A., A. El Marrakchi, F. 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