Virulence Genes and Antibiotic Resistance Pattern of Yersinia Entrocolitica Isolated from Raw Milk and Traditional Cheeses

Pathogenic microorganisms are one of the important factors in contaminating and endangering the safety of foods for consumers, and continuous control of foods in terms of the presence of pathogenic microorganisms can guarantee the health of foods. Yersinia entrocolitica is a foodborne pathogenic bacterium. Raw milk and fresh traditional cheese may be contaminated with this bacterium. Generally, Iranians tend to consume traditional and homemade foods more than industrial products. This issue is especially relevant to milk products such as cheese, butter, and local cream. The purpose of this study was to determine the contamination of raw milk and traditional cheeses produced in Chaharmahal and Bakhtiari province with Yersinia entrocolotica.

A total, of 100 raw milk and 50 traditional cheese samples were obtained randomly from dairy plants and milk collection stations, as well as retail shops. The initial enrichment of the samples was performed in the TSB medium. Then, 100 microliters of the medium was centrifuged and 25 microliters of its sediment was cultured on the special medium of Cefsoludin Irgazan Novobiocin agar (CIN) with supplement (containing Cefsoludin 7.5 mg, Irgasan 0.2 mg and Novobiocin 1.25 mg). Suspicious colonies (red colonies) were identified on the CIN agar and biochemical tests including IMViC, Ureas, and H2S production.DNA extraction was performed by boiling method and polymerase chain reaction (PCR) was performed for the identification of Ali and Yst genes using specific primers (Table 1). Table 1. Characteristics of Primers Used to Identify Yersinia Enterocolitica Genes Antibiotic resistance test was performed by the disk diffusion method on Muller-Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI). Resistance of Yersinia enterocolitica isolates to 7 commonly used antibiotics in treatment including cefixime (5µg), gentamicin (10µg), tryptoprim (5µg), ciprofloxacin (5µg), amoxicillin (10µg), tetracycline (30µg), and sulfamethoxazole (300µg).

Out of 100 samples of raw milk and 50 samples of traditional cheese, 20 samples (13.3%) suspected Yersinia enterocolitica bacteria were isolated. of these, 14 samples (14%) were related to raw milk samples and 6 samples (12%) were related to traditional cheese samples. The results of the PCR test showed that 5 isolates (3.3%) were Y. enterocolitica bacteria, 4 isolates (4%) related to raw milk, and 1 isolate (2%) related to traditional cheese. In addition, in the evaluation of virulence genes, it was found that out of 4 raw milk isolates, 1 isolate carried the Yst gene, 2 isolates carried the Ail gene, and 1 isolate had both genes. Also, 1 isolate of Y. enterocolitica isolated from traditional cheeses carried the Ail gene (Table 2). Table 2. Percentage of Contamination of Raw Milk and Traditional Cheese with Yersinia entrocolitica, (%) The results of antibiogram tests on Yersinia enterocolitica isolates showed the highest resistan.ce to Gentamicin, Trimethoprim, Ciprofloxacin, and Sulfamethoxazole antibiotics, and moderate resistance to cefixime and amoxicillin (Table 3).  Table 3. Antibiotic Resistance Pattern of Yersinia Entrocolitica Isolated from Raw Milk and Traditional Cheese.

In general, the results of the present study and other studies in Iran and the world show that raw milk and traditional cheeses, contaminated with Yersinia enterocolitica, which carry virulence genes and are resistant to some common antibiotics, can endanger the health of consumers.