[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.

Growth and Persistence of Campylobacter jejuni in Foodstuffs.

Campylobacter genus bacteria causing campylobacteriasis are difficult to culture. This fact necessitates creation of special approaches to studies of the behavior of these pathogens during the manufacture and storage of foodstuffs. The regularities of Campylobacter jejuni transition into an uncultivable state are studied under conditions simulating the process of immersion cooling of fresh poultry products. The proportion of viable colony-forming (CFU) cells to the total count of planktonic and uncultivable cells in the population was calculated by the level of genomic DNA in the samples evaluated by quantitative real-time PCR with intercalating dyes. PCR was carried out with primers detecting the cytolethal toxin subunit B gene cdtB and invasion gene ciaB in C. jejuni strains. The count of detected cells was 5-10-fold higher than the count of CFU; the cultural method failed to detect the agent in 40% analyzed samples of superficially infected products, while the level of uncultivable cells detected by PCR was significantly higher. The relationship between culturing conditions and formation of C. jejuni biofilms was studied. The most intensive formation of film exomatrix was observed under unfavorable conditions for this microorganism at 25oC. In microaerophilic gaseous medium, weak formation of films and intensive growth of C. jejuni populations were observed. Culturing at higher temperatures (37-42oC) was in fact inessential for the film formation process.

[Subacute oral toxicity in vivo of multi-walled carbon nanotubes].

Multiwall carbon nanotubes (MWCNTs) are regarded as environmental pollutants with increased risk. Recently MWCNTs have attracted attention as a promising component of packaging materials for food products, as carriers for agricultural plant growth stimulants, agrochemicals components and advanced pesticides, which creates the possibility of their exposure through the gastrointestinal tract. Objective of the research is assessment of sub-acute oral toxicity to rats of MWСNTs in an experiment lasting 100 days. MWCNTs preparation «Taunit-M®¼ was preliminary characterized by electron microscopy and Raman light scattering. Nanomaterial was administered to animals as sonicated dispersion in water with 1% by weight of nonionic surfactant Tween 20. The experiment was performed on 80 growing male Wistar rats with initial body weight (b.w.) 86±2 g. Rats of experimental groups (from 2nd to 5th) received MWCNTs dispersion instead of drinking water, the animals of the 1st control group - a carrier solution (Tween 20). Doses of MWCNTs consumed were, respectively, in groups 1-5: 0; 0.01; 0.1; 1.0 and 10 mg/kg b.w. Hematological and biochemical indices of blood were determined together with the activity of glutathione peroxidase of erythrocytes, the content of non-protein thiols in the liver, excretion of 8-hydroxy-2'-deoxyguanosine (8-oxo-G) and content of the main and transient components of gastrointestinal microbiocenosis in the cecum contents. Apoptosis of hepatocytes was studied by flow cytometry. As a result MWCNTs led to increase of blood glucose and creatinine in rats in group 2, a significant decrease in the number of neutrophils and monocytes by increasing the number of lymphocytes, decreased platelet volume, the most pronounced also in group 2, receiving the lowest dose of MWCNTs. There were no signs of oxidative DNA damage identified. At the lowest dose MWCNTs caused a significant decrease in the number of bifidobacteria, increase - citrate-assimilating Enterobacteriaceae, hemolytic aerobic microorganisms and yeast. These changes in the microbiota should be considered as adverse, apparently leading to disturbances in the immune function.

[Optimization of microbiological methods for food control based on the differential-diagnostic media for the isolation and cultivation of bacteria of the genus Campylobacter].

А screening study on the detection of campylobacteria in raw food products, semi-finished products and objects in the external environment in the poultry processing industry was conducted. The highest level of detection of campylobacteria is set for raw poultry products, including carcasses of broilers, turkeys and quail. A general accordance of getting Campylobacter in raw materials and food products with inadequate sanitary treatment of separate areas of production has been established: in most cases Campylobacter spp. was extracted from the samples, contaminated with coliform bacteria and Salmonella. It is shown that the frequency of contamination of raw poultry with pathogens is largely dependent on the cooling of the carcasses. When using the immersion method, the conditions for cross contamination with pathogens through water bath cooling are present (45% of samples infected with Campylobacter spp.). Under combined use of super-cooled water and aerosols Campylobacter were also detected quite often in 27% of samples. Contamination by pathogens was the lowest in evaporative cooling method with the use of antimicrobial hydrospray (less than 5% positive samples), allowing to recognize this method as the most promising for the production of microbiological safe products. The work on optimization of nutrient medium composition and adaptation recommended methodological scheme of analysis for detection and species identification of bacteria of the genus Campylobacter was carried out. Formulation of traditionally used growth media was modified, and balanced composition of growth and selective components was matched in accordance with the requirements of existing standards. Given the urgency of increasing the effectiveness of the methods of control of campylobacteria in foods and the lack of domestic analogues of the culture media in the Russian Federation, an optimized method for the production of dry nutrient media for the detection, identification and storage of campylobacteria isolated from food products and clinical material was developed. The conducted study allowed to develop Technical conditions 21.20.23-006-01897222-2016 «Dry culture medium for detection of bacteria of the genus Campylobacter¼ and «Instruction for use of culture media¼. Depending on the purpose of the medium produced in the following versions: the selective broth for enrichment of campylobacteria; differential selective agar for isolating and quantifying of Campylobacter spp.; semi-solid nutrient agar for cryostorage of Campylobacter strains. The list of criteria for assessing the quality of commercially available lots of dry media included: solubility, pH, gel strength of agar, the content of amine nitrogen, specificity, selectivity, growth and inhibitory properties. The practical application of these media in terms of the national laboratories will significantly simplify the use of existing standards developed based on international ISO standards, but not adapted to the main range of commercial media and reagents used in routine food control for the presence of campylobacteria.

[Antibiotic resistance of Campylobacter jejuni strains isolated from food products].

Campylobacter jejuni is a leading member of the genus Campylobacter which cause up to 90% of laboratory confirmed cases of campylobacteriosis. The most important characteristic defining the biological features of C. jejuni is their sensitivity to antibiotics. Agricultural intensification, expansion of the range of the used disinfectants and antiseptics, uncontrolled use of antibiotics in animal production is increasingly leading to the selection of the most resistant forms of Campylobacter with antibiotic resistance and multiple virulence factors. The study of antibiotic resistance of C. jejuni isolated from food and environment need for the development of new approaches for laboratory diagnosis of campylobacteriosis and confirmation of the role of food path of transmission, for creation the system of preventive measures to reduce the risk of contamination of food by Campylobacter spp. in Russia. The aim of this study was to investigate the phenotypic profiles of antibiotic resistance of Campylobacter spp. isolated from poultry and the environment in the poultry processing industry. In the analysis of 110 samples of raw poultry products and swabs from surfaces of the equipment 55 strains of the genus Campylobacter were selected, including 46 strains of C. jejuni. For study sensitivity of these strains to 15 antimicrobials (8 classes) disk diffusion assays were done using the EUCAST protocol. The following antibiotics were used: nalidixic acid, ciprofloxacin, erythromycin, gentamicin, amikacin, kanamycin, tetracycline, oxytetracycline, doxycycline, clindamycin, lincomycin, ampicillin, chloramphenicol, florfenicol, cefotaxime. All C. jejuni strains were resistant to cephalothin, which confirms their belonging to this species. 89% of the strains were insensitive to nalidixic acid, which indicates the reduction of informativeness of this test, traditionally used in the standard scheme of species identification of Саmpylobacter spp. Most of the investigated isolates were resistant to ciprofloxacin (96.3%) and tetracycline (88.6%), 34% of strains had resistance to erythromycin; 40% of tested C. jejuni were multi-resistant to four or more antibiotics. The data indicate a high prevalence of antibiotic-resistant strains among campylobacteria, contaminated poultry products during the processing of raw materials.

[The study of the contamination and the levels of Campylobacter spp. during the processing of selected types of foods].

The purpose of the work was to study the nature of the Campylobacter spp. contamination during the processing of food products of plant and animal origin (raw poultry and beef meat, raw milk, leafy salads, sliced raw vegetables). In the study of 148 samples 50 strains of Campylobacter spp. (33.8%) were found. For the main phenotypic characteristics they were identified as C. jejuni spp. jejuni and C. jejuni spp. doylei (over 75%). The highest level of detection of campylobacteria (over 45%) was set for raw poultry, including the carcasses of chickens broilers, quails, turkeys and their semi-finished products. 19 of the 27 strains from poultry were identified as C. jejuni. Among the strains isolated from the environment, including swabs from equipment surfaces, 91% of the isolates were also presented by C. jejuni. It was found that the investigated foodstuffs were characterized by high levels of contamination with bacteria of the family Enterobacteriaceae, the content of which was comparable with the identified values of total viable bacteria (cfu). Salmonella was detected in 19% of the investigated poultry samples and in 14.3% of raw cow milk. In the study of swabs from surfaces of poultry processing equipment, the frequency of detection of Campylobacter strains was 38.7%, Salmonella - 12.9%. Most commonly Campylobacter and Salmonella were detected in the zones of primary processing of poultry: the frequency of isolation of Salmonella in slaughter corner was 25%, Campylobacter - 43%. When testing the swabs taken in the cooking zone of «fast food¼ restaurants Campylobacter and Salmonella were not detected. For studying the swabs from equipment surfaces and the environment for the presence of Campylobacter spp. a modified technique of sampling was developed. The method includes a comprehensive analysis in the test area with the use of three types of media for transportation and incubation of Campylobacter spp. (Preston broth with blood, Brucella broth, Cary-Blair medium), that increase the probability of detection of these pathogens.

[Changes in the phase variant spectra in the populations of lactic acid bacteria under antibiotic treatment].

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

[Mechanisms of hemopoiesis inhibition and recovery after the administration of carminomycin and rubomycin in an experiment]. / Mekhanizmy podavleniia i reparatsii gemopoéza pri vvedenii karminomitsina i rubomitsina v éksperimente.

The experiments on 766 albino non-inbred rats and 270 mice BALB/c showed that when there were no basic differences in the mechanism of the anthracycline myeloinhibitory effect associated with a decrease in the number of the exogenic trunk hemopoietic cells, inhibition of the bone marrow proliferative activity and a decreased rate of the hemopoietic elements differentiation, carminomycin induced a more pronounced and persisting impairment of the hematopoietic tissue proliferation, as compared to rubomycin on their administration in doses equivalent by the total toxicity.

[Effect of carminomycin on bone marrow cell proliferative activity and differentiation rate]. / Vliianie karminomitsina na proliferativnuiu aktivnost' i skorost' differentsirovki kletok kostnogo mozga.

Autoradiographic investigations with the use of 3H-thymidine were performed on 140 noninbred albino rats. The fate of the label in the cells of the myeloid series was followed in the experimental and control groups. The animals of the experimental group were treated with carminomycin in the LD50 30 minutes after injecion of 3H-thymidine. The mitotic cycle of the nondifferentiated cells and promyelocytes + myelocytes in the control animals was equal to 24 hours, the period and rate of the metamyelocyte maturation being 10 hours and 0.81 per cent/hour respectively. The mitotic cycle of the nondifferentiated cells and promyelocytes + myelocytes during the first division after carminomycin administration increased up to 48 hours due to the block of G2-period, the period and rate of the metamyelocyte maturation being 18 hours and 0.43 per cent/hour respectively. The myelotoxic effect of carminomycin was due to the damage of the dividing cells in G2-period of the mitotic cycle, decreased rate of differentiation and death of the cells after or during the first and second divisions.

[Mechanism of the myeloinhibitory effect of carminomycin]. / K mekhanizum mieloingibiruiushchego éffekta karminomitsina.

The proliferative activity and level of aberrant mitoses in the cells of the bone marrow were studied experimentally on 223 noninbred mice treated with carminomycin administered intraperitoneally in single (LD50) and repeated doses. When the antibiotic was used in a single dose the values of the mitotic activity of the bone marrow elements did not correspond to the severity of depression and thir quantitative composition, which was explained by an impairement of the mitosis quality and possible interkinetic destruction of a significant part of both erythroid and immature myeloid cells capable of division at early stages after the exposure. At the same time the level of the bone marrow devastation under conditions of the treatment with repeated doses was mainly determined by inhibition of the erythronormoblast proliferative activity.