Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Improvement of Methods for Safety Control of Microbial Producers and Food Produced Therewith.

The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.

[Mycotoxins contamination of cocoa products and carob marketed in the Russian Federation].

Cocoa is a daily basic food for many people all over the world. Also, people engaged in healthy lifestyle often prefer carob. The aim of the present study was to evaluate occurrence of Aspergillus, Penicillium, Fusarium and Alternaria secondary metabolites in cocoa and carob available in the Russian Federation and assess mycotoxin's intake with these products. Material and methods. Concentration of 27 mycotoxins in 63 samples of cocoa and carob products was determined by ultra high-performance liquid chromatography coupled to tandem mass-spectrometric detection (UPLC-MS/MS). The list of mycotoxins included regulated ones (aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, T-2 toxin, zearalenone), their derivatives and structural analogs (A and B trichothecenes), and emergent mycotoxins (alternariol, its monomethyl ether, altenuene, tentoxin, citrinin, sterigmatocistin, cyclopiazonic and mycophenolic acids, enniatins, beauvericin). Results. 29 of 41 cocoa samples were positive for beauvericin, contamination level was from 1.6 to 2184.8 µg/kg. Tentoxin, sterigmatocystin and alternatiol monomethyl ether were detected in 3 samples; their concentration varied in the ranges 0.7-1.2, 1.5-3.3 and 4.0-7.8 µg/kg correspondingly. Carob samples were positive for alternariol (14 of 22 samples, 1.5-43.1 µg/kg); tentoxin (13 samples, 0.5-8.7 µg/kg), mycophenolic acid (6 samples, 6.9-8.2 µg/kg) and for alternatiol monomethyl ether (3 samples, 1.0-1.2 µg/kg). Several samples of cocoa were contaminated with regulated mycotoxins: zearalenone (18 of 41 samples, 2.1-24.6 µg/kg), ochratoxin A (14 samples, 0.75-12.0 µg/kg) and aflatoxin B1 (2 samples, 0.59 and 0.86 µg/ kg). Several carob samples were contaminated with fumonisin B2 (7 of 22 samples, 4.2-5.2 µg/kg), ochratoxin A (5 samples, 0.5-1.4 µg/kg) and aflatoxin B1 (3 samples, 0.15-0.18 µg/kg). The ochratoxin A content in two instant cocoa beverages exceeded its maximum level (5 µg/kg) set for some plant products. Conclusion. To the best of our knowledge, the present study is the first survey devoted to emergent mycotoxin contamination of carob and cocoa alternative products marketed in the Russian Federation. The high occurrence of such mycotoxins as aflatoxins, beauvericin and ochratoxin A in these products indicates a potential health risk and the need for a hygienic assessment of cocoa and carob products' contamination not only with regulated in cocoa products aflatoxin B1, but with other mycotoxin including emergent ones. The risk of dietary intake of aflatoxins, beauvericin and ochratoxin A with cocoa products (especially with instant cocoa beverages) has been revealed for children over 7 years old in organized groups.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

Bacterial Pathogens in Food: Modern Safety Criteria and Methods of Molecular Genetic Analysis.

The review is devoted to the issues of improving the food biosafety system by developing and implementing modern methods of microbiological and molecular genetic analysis and new safety standards for the main groups of food products. The results of fundamental and applied microbiological research in the field of food safety conducted by the Federal Research Center for Nutrition and Biotechnology are presented. We demonstrated the necessity of a detailed study of the ecology and features of survival of new types of pathogenic microorganisms and evaluation of the role of technological factors in the formation of altered properties of these pathogens in food production environment. It is noted that the most effective basis for establishing criteria for biosafety of food products is the use of a structural model of microbiological risk analysis (MRA), which implies assessment and integration of risks throughout the food chain and step-by-step study of microbial hazards, predicts the degree of danger to the health of certain foods contaminated by them and the risk and severity of adverse effects. Consideration of the variability of pathogens and the ability of MRA to forecast allows rationalizing resources in the most relevant areas when developing measures to protect the population. It is shown how, as a result of studying the biology of pathogenic bacteria of the genera Campylobacter, Salmonella, Cronobacter, and Listeria and mechanisms for regulating the expression of pathogenicity and resistance factors of these pathogens, improved schemes and modified methods have been developed over a number of years, allowing accelerated indication of pathogens in food products using combined schemes of bacteriological and molecular genetic analysis based on the selection of the most informative biochemical and immunological tests, and their genotyping. Accumulated scientific data in the field of biosafety indicate the need for further research in the prediction of the pathogen behavior in food, studying the mechanisms of regulation of the expression of pathogenicity genes in food-borne pathogens, the transmission of antibiotic resistance genes, and studying the interaction of the human with pathogens to calculate the biological response to microbial agents coming from food.

[Prospective molecular methods for sequencing microorganisms in the system of assessment and control of food safety].

To solve one of the most important tasks of food hygiene - ensuring foodstuff biosafety, the leading place is given to the use of nucleic acid sequencing methods that allow to identify and describe the nucleotide sequences of genomes both in individual microorganisms and metagenomic characteristics of microbial communities in environmental objects, in humans and animals. The purpose of the review was to analyze the main areas of sequencing application in food microbiology, biotechnology and epidemiology. Material and methods. The collection and analysis of scientific and informational materials published in domestic and foreign publications from Scopus, Web of Science, PubMed, RSCI databases, official reports of the World Health Organization (WHO), the European Food Safety Agency (EFSA) and other published sources has been carried out. Results. The review presents generalized characteristics of the most well-known sequencing formats based on different principles of reading and processing genetic information: capillary sequencing by Sanger, pyrosequencing, sequencing by ligation, sequencing by DNA synthesis, semiconductor high-performance sequencing, sequencing of individual DNA molecules. It is shown that multilocus analysis (MLST, MLVA) increases the specificity of genotyping and intraspecific identification of food-borne pathogens. It is used to assess the pathogenic potential and study the mechanisms of evolutionary variability of bacterial pathogens, leading to polymorphism of clonal lines and the appearance of strain-specific differences, to monitor the different genotypes of pathogens in production and other objects of the food chain. Nanopore sequencing is promising for studying bacterial plasmids and localization of resistance genes of MDR bacteria. Genome-wide analysis (WGS) makes it possible to identify detailed characteristics of pathogens of infectious diseases, including uncultivated and previously unknown microorganisms, as well as to study the features of the organization of individual pro- and eukaryotic genomes, gene regulation systems, pathogenicity factors and protein expression. The role of WGS methods for determining the degree of genetic kinship of infectious agents isolated from various sources in the investigation of outbreaks and sporadic food poisoning and infections is shown. The possibilities of metagenomic sequencing in the study of species composition, genetic diversity and metabolic potential of microbial communities are demonstrated. The main advantage of metagenomics is the possibility of direct sequencing of the genomes of microorganisms living in the human body, animals, water, soil and other objects. Metagenomic analysis plays a leading role in the detection of unexplored taxa, uncultivated and difficult-to-cultivate forms of a wide range of microorganisms. The most important direction of metagenomics is the study of the human or animal microbiome, the analysis of the relationship between the intestinal microbiota and metabolism, as well as the formation of the nutritional status of a person. Theoretical and practical aspects of the implementation of high-performance sequencing methods in the system of food biosafety assessment and control are reviewed: safety assessment of genetically modified strains of food producers, development of fundamentally new algorithms for investigating outbreaks of infectious diseases, creation of network systems for early diagnosis and notification of food infections of bacterial and viral etiology. Conclusion. Sequencing should become a standard methodology in the field of food safety for the identification and characterization of food pathogens, including those with resistance to antibiotics and adverse technological effects. The introduction of WGS into the food safety assessment and control system will make it possible to assess real food contamination with new and newly returning pathogens, create electronic databases and scientifically substantiate the most adequate measures for the prevention of food infections and ensuring the health of the population.

[Gut microbiome: from the reference of the norm to pathology].

А systemic assessment of the state of the human intestinal microbiome was carried out in relation to its function in the macroorganism, aimed at providing the nutriome, and the factors that determine the adequate nutritional status. A new concept of "reference gut microbiome of a healthy person" was postulated and the requirements to it were formulated: interaction with the host according to the principle of mutualism, provision of immune balance with the macroorganism due to the correct formation of mucosal immunity, implementation of metabolic and regulatory functions without losses for the nutriome. A set of characteristics and biomarkers reflecting the taxonomic composition and population properties of the microbial community, as well as the state of its essential immune and metabolic functions, was proposed as a criterion for its assessment in healthy adults who consume a diet balanced in nutritive and energy value, appropriate for age and energy spending. The influence of alimentary factors on the formation of the human intestinal microbiome in early ontogenesis, the nature of dysbiotic shifts, including those under common non-infectious alimentary-dependent diseases (obesity, food allergy, urolithiasis), in Russians were studied, the ways of their correction and maintenance of the intestinal microbiota in the process of life were substantiated taking into account modern knowledge.

[Microbiological safety of food: development of normative and methodical base].

The main results and prospects of fundamental and applied hygienic research of the laboratory of biosafety and nutrimicrobiome analysis of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (hereinafter - the Institute of Nutrition) in the direction of developing a regulatory and methodological framework for assessing the microbiological safety of food are reviewed. The formation of microbiological regulation as a scientific analytical and administrative managerial process in the former USSR and the Russian Federation is considered in the context of historical data, including personal contribution of the scientists of the Institute of Nutrition and other specialists. The basic principles of regulation are emphasized: the scientific validity of the established criteria and requirements, the feasibility, technological attainability, differentiation according to the degree of danger to the health of consumers, preventive nature. The resource of the national normative and methodological base in the field of microbiological food safety at the turn of the century is characterized, the features of the introduction of the microbiological risk assessment (MRA) methodology in the substantiation of Russian norms and measures for the prevention of food infections are described. The information is given on the developed guidance documents on MRA and on the examples of norms adopted on its basis. The article covers the issues of reglamentation the requirements for food safety and reducing the spread of new pathogens Stx-Escherichia coli, Listeria monocytogenes, Enterobacter sakazakii, Campylobacter spp. in the food chain based on risk-oriented approaches. The necessity of taking specific measures for the prevention of cross-contamination in the poultry processing industry is substantiated, taking into account the evidence of the high adaptability of C. jejuni isolated from domestic raw poultry. In the sanitarian-mycological aspect, the monitoring perspective of mould fungi, taking into account their chemotypes, in cereals and non-grain plant products is shown to predict the risk of mycotoxin accumulation and take timely measures. The need to assess the impact on the population, taking into account the characteristics of consumption in the country, as well as the development of criteria for indirect risk of residues are argued for regulation of the antibiotics in food. In light of the challenges in the field of agro- and food technologies to public health at the present stage, contributing to the acceleration of microbial evolution and the emergence of new risks in food, the priority tasks of improving the regulatory and methodological base for assessing microbiological safety have been identified, with an emphasis on the introduction into the process of substantiating the norms of innovative OMICs-technologies based on the achievements of genomics, transcriptomics, proteomics, metabolomics, bioinformatics.

Formation of the Resistance of Campylobacter jejuni to Macrolide Antibiotics.

The tendency to the formation of macrolide resistance in campylobacteriosis pathogens is considered as a serious threat to public health due to ubiquity of campylobacter strains resistant to a wide range of antibiotics, primarily fluoroquinolones and tetracyclines. To assess the prevalence of resistant Campylobacter spp., we performed screening for macrolide sensitivity among 40 Campylobacter jejuni strains isolated from raw milk, poultry product, and washings from the equipment of the poultry processing plants. Phenotypic resistance to erythromycin, the most popular antibiotic for the treatment of campylobacteriosis, was revealed in 27.5% C. jejuni strains; 10% strains were resistant to azithromycin. The search and selection for gene markers of Campylobacter resistance to macrolides was performed. It was found that the resistance of C. jejuni to erythromycin is realized mainly via synthesis of proteins that protect ribosomes (the presence of coding sequences was detected in 45% of the studied strains) and the transmembrane pump mechanism (efflux pump CmeABC genes were found in 36% isolates); both mechanisms are transmissible. Chromosomal mutations in the 23S rRNA sequence detected in 18% strains seem to play a less significant role.

[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.

Peculiarities of Biofilms Formation by Campylobacter Bacteria in Mixed Populations with Other Microbial Contaminants of Food Products.

Peculiarities of biofilms formation by Campylobacter bacteria in mixed populations with other microbial contaminants was studied by real-time impedance spectroscopy on an automated xCelligence real time cell analyzer (RTCA). This method is based on measuring the medium resistance in special plates (E-plates) with interdigitated microelectrodes. Coculturing of campylobacter with coliform bacteria is accompanied by film formation; the intensity of this process varies depending on the type of the test cultures and the nature of their interaction in mixed populations. Film formation by C. jejuni during co-culturing with enterobacteria is maximum during the first hours and depends on the presence of stress factors in the environment. The biomatrix film was synthesized by 3 times more intensively in the presence of oxygen than in microaerobic conditions, and also by 1.7-4.3 times more active in the mixed culture with Enterobacter cloacae, E. coli, and K. pneumoniae. During co-culturing of campylobacter with salmonella, no enhanced film formation by the tested strains was observed. Unlike members of the genus Enterobacter intensively producing exopolysaccharides, pathogenic member of Enterobacteriaceae, salmonella, demonstrated weak capacity to form film matrix. The study of film formation by Campylobacter allows more accurate assessment of the effectiveness of sanitary bactericidal treatment of food industry facilities, predict the appearance of biofilms and the intensity of their formation depending son the nature of the antimicrobial effect and the used means.

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.

Studying the Phenotypic and Genotypic Expression of Antibiotic Resistance in Campylobacter jejuni under Stress Conditions.

Specific features for the development of resistance in Campylobacter jejuni strains were studied after treatment with antibiotics of 6 pharmacological groups. Populations of 18 native strains of C. jejuni (isolated from raw poultry products) and their subcultures (obtained after 2-3-fold stress exposures to antimicrobial agents in subinhibitory doses) were examined to evaluate the expression of phenotypic antibiotic resistance. Genotypic properties of strains were studied by the PCR with primers that detect the presence of genes for resistance to aminoglycosides (aphA-1, aphA-3, and aphA-7), tetracyclines (tetO), and quinolones (GZgyrA). The majority of test strains of C. jejuni exhibited a high resistance to nalidixic acid, ciprofloxacin, and tetracycline, which reached the maximum value after numerous passages. The expression of antibiotic resistance was greatest in the presence of nalidixic acid and tetracycline. Ciprofloxacin resistance of 33% strains, which were initially resistant to this antibiotic, was increased after 2-3-fold treatment. We revealed a high degree of correspondence between phenotypic and genotypic profiles of antibiotic resistance in food isolates of Campylobacter. One, two, or more genes of aphA were identified in 85% strains phenotypically resistant to aminoglycosides. The tetO gene was found nearly in all strains resistant to tetracycline. Studying the biofilm matrix in C. jejuni after culturing with antibiotics in subinhibitory doses showed that quinolones (particularly nalidixic acid) and tetracyclines potentiate the formation of biofilms and increase the tolerance of Campylobacter to stress exposures. The intensity of biofilm growth was shown to depend little on the effect of macrolides and aminoglycosides. Therefore, the presence of these agents in residual concentrations is associated with a lower risk for the development of antibiotic resistance in C. jejuni populations.

In Vitro Culturing and Storage of Campylobacter Genus Bacteria.

Experimental model for in vitro evaluation of Campylobacter genus bacteria growth kinetics, inhibition, or inactivation is proposed. The model allows quantitative evaluation of the sensitivity to various types of stress exposure and promotes detection of the regularities of their transformation into uncultivable forms. The model implies the use of 96-well plates for parallel culturing of several subpopulations of the test strain in media with various parameters. The proposed algorithm includes evaluation of the proportion of viable CFU to total level of planktonic and uncultivable cells in the population, which is estimated by the content of genomic DNA in the samples by quantitative PCR (or real-time PCR) with ciaB, cdtB, or 16S rRNA primers. The presence of biofilm matrix is detected by the intensity of staining of polystyrene plates. This model can be used for evaluation of the most significant types of exposure, including low-dose antibacterial treatment, promoting the formation of stable microorganism variants. The model has been used to study the effects of culturing conditions on the characteristics of C. jejuni populations. The most characteristic feature of C. jejuni is reduction of the count of viable cells up to complete disappearance of cultivable forms under favorable conditions of growth. The level of viable cells in the populations decreased 10-fold and more, on average, after 48-h incubation. Not all strains exhibit this property, some strains retain their viability, which is detected by the culturing method, and contributes to biofilm formation.

Formation of Biofilms by Foodborne Pathogens and Development of Laboratory In Vitro Model for the Study of Campylobacter Genus Bacteria Based on These Biofilms.

We analyzed the formation of biofilms by 7 strains of Campylobacter genus bacteria and 18 strains of Enterobacteriaceae genus bacteria that were isolated from plant and animal raw materials, from finished products, and swabs from the equipment of the food industry. Biofilm formation on glass plates, slides and coverslips, microtubes made of polymeric materials and Petri dishes, and polystyrene plates of different profiles were analyzed. When studying the process of films formation, different effects on bacterial populations were simulated, including variation of growth factor composition of culture media, technique of creating of anaerobiosis, and biocide treatment (active chlorine solutions in a concentration of 100 mg/dm3). The formation of biofilms by the studied cultures was assessed by the formation of extracellular matrix stained with aniline dyes on glass and polystyrene surfaces after incubation; 0.1% crystal violet solution was used as the dye. The presence and density of biomatrix were assessed by staining intensity of the surfaces of contact with broth cultures or by optical density of the stained inoculum on a spectrophotometer. Biofilms were formed by 57% Campylobacter strains and 44% Enterobacteriaceae strains. The intensity of the film formation depended on culturing conditions and protocols, species and genus of studied isolates, and largely on adhesion properties of abiotic surfaces. In 30% of Enterobacteriaceae strains, the biofilm formation capacity tended to increase under the influence of chlorine-containing biocide solutions. Thus, we developed and tested under laboratory conditions a plate version of in vitro chromogenic model for evaluation of biofilm formation capacity of C. jejuni strains and studied stress responses to negative environmental factors.

[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 influence of stresses on virulence genes expression in foodborne pathogens Campylobacter jejuni].

The study of the responses to cold exposure in Campylobacterjejuni (C. jejuni)--one of the most common foodborne pathogens is important for elucidating the mechanisms of acquisition of products contaminated with campylobacter, hazardous properties. These data are also necessary to create effective systems of microbiological controls at all stages of production and storage of food. 5 pairs of oligonucleotide primers were selected for detecting of genes cadF, cdtB, ciaB, flaA, iamA, encoding the main factors of pathogenicity of foodborne pathogens Campylobacter jejuni--adhesion and invasion of epithelial cells, production of CDT-toxin and mobility. To quantify the expression levels of target genes of C. jejuni a comparative method of determining the amount of amplification products of genes encoding pathogenicity factors of Campylobacter spp. has been developed using real-time PCR with intercalating dyes. To calculate and quantify gene expression the mathematical models have been obtained that allow extrapolation of threshold cycles of amplification to the initial number of copies of RNA/DNA in the tested samples. It has been established that exposure of C. jejuni at low temperatures +4 degrees C did not lead to increased levels of expression of genes cdtB and ciaB. However, in the populations of C. jejuni subjected to freezing, followed by incubation at optimum for the pathogen temperature of +42 degrees C, the increase in expression of mRNA encoding protein subunit B of CDT-toxin and antigenic marker of invasion took place. The number of copies of RNA in C. jejuni after stress exposure increased by 1.14-2.6 lg in comparison with intact cultures. CdtB and ciaB gene expression in C. jejuni can serve as an indicator of cell response to stress and helps to restore the functions of the bacterial cells after the termination of cold exposure and return of the pathogen in conditions favourable to the realization of its pathogenic potential.

[Toxicological evaluation of colloidal nano-sized silver stabilized polyvinylpyrrolidone. IV. Influence on intestinal microbiota, immune indexes].

Nano-sized colloidal silver (NCS) stabilized with polyvinylpyrrolidone (PVP) containing nanoparticles (NPs) of silver with a diameter of 10-80 nm was administered to growing male rats (body weight 80±10 g) during the first 30 days by intragastric gavage and then for 62 days with diet consumed in doses of 0.1, 1.0 and 10 mg/kg of body weight per day based on silver (Ag). The control animals received deionized water and PVP. The composition of microbiota from the cecum was studied using standard microbiological methods with determination of the main and transient components, together with antagonistic activity of symbiotic bifidobacteria. Expression of antigens CD45RA, CD3, CD4, CD8, CD161a on lymphocytes (Ly) of peripheral blood was determined by flow cytometry; blood serum levels of cytokines IL10, IL13, TNFα were examined by ELISA. It was shown that subacute administration of colloidal Ag in all studied doses did not lead to significant changes in the composition of the main components of normal microbiota, providing, however, the inhibitory effect on the growth of some transitory components probably including opportunistic species of microorganisms. Among the studied immunological parameters decreased amount of B-Ly was noticed at the highest dose of the NCS, while changes in the other parameters of the immune system were depended ambiguously on the dose of the product. The results were analyzed in conjunction with the data of previous publications concerning the impact on the NCS on integrated, morphological, hematological, biochemical and enzymological indexes of animals in the 92-day experiment. It was concluded that significant symptoms of NCS sub-acute oral toxicity manifested starting from a dose of 1 mg/kg body weight of Ag, and the maximum not observed adverse effect dose (NOAEL) can be estimated as 0.1 mg/kg body weight.

[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.