[The study of the raw milk contamination by bacteria of the genus Campylobacter using traditional microbiological methods and quantitative PCR assay].

Gastroenterocolitis caused by Campylobacter bacteria are the most common acute infectious zoonotic foodborne diseases. One of the important factors for the transmission of infection is contaminated dairy products, so the assessment of contamination of raw milk with Campylobacter is necessary to develop effective measures to suppress the growth of the pathogen and ensure the safety of products. The aim of the study was to assess the microbial characteristics of raw milk samples and the nature of their contamination with thermophilic bacteria of the Campylobacter genus. Material and methods. A total of 60 samples of raw milk from the central regions of the Russian Federation and 48 experimentally infected samples of raw milk were studied. To assess the microbial contamination of milk, the number of extraneous microflora was determined, including coliform bacteria (CFB). The identification and quantification of bacteria of the genus Campylobacter was carried out by cultural methods in comparison with quantitative PCR assay. For PCR, primers were used that detected the speciesspecific sequence of C. jejuni 16s rRNA, the presence of the cytotoxic toxin gene cdtB and the invasion gene ciaB. Results and discussion. A significant part of the samples of raw milk (31.6%) was characterized by high levels of microbial contamination exceeding 106 CFU/cm3. Gram-negative bacteria were the dominant type of bacterial microflora, their levels were comparable with the detected values of the total number of microorganisms. Coliform bacteria were found in all studied samples, and their content in 90% of the samples reached 105 CFU/cm3, and in some samples - 107 CFU/cm3. Campylobacter spp. detection rate in raw milk was 8.3%, and their number ranged from 0.1 to 100 CFU/cm3 (average 2.0×10 CFU/cm3). The isolated strains of campylobacters were identified as a C. jejuni species. In the study of the microbial background of the examined samples of raw milk, a comparative analysis of their contamination by campylobacters by rti-PCR was simultaneously carried out. The majority of samples (over 60%) were positive for the presence of 16s rRNA genomic sequence, and they were characterized by the highest values of total bacterial contamination and the amount of coliforms. The use of a multi-primer approach (simultaneous testing for the presence of 16s rRNA and the gene of cytoletal toxin cdtB C. jejuni) reduced the number of positive cases of Campylobacter DNA detection to 16.6%, which suggests a greater informative value of the cdtB gene for the detection of viable, including uncultivated cells. An indicative assessment of the results in a quantitative format showed levels of 104-106.5 genomic equivalents of the DNA in 1 cm3, suggesting the possible presence of viable Campylobacter cells in the tested probes with a significantly greater frequency than that established by cultural method. Conclusion. At low levels of Сampylobacter contamination the microbiological methods do not provide reliable detection of the pathogen due to massive contamination of raw milk by extraneous microflora. Campylobacter spp. were detected by the culture method in 8.3% of cases, while the use of multi-primer PCR assay with cdtB and ciaB genes can double the detection of C. jejuni in raw milk samples.