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.

[The study of the content of short-chain fatty acids in the intestine of people with lipid metabolism disorders].

Dyslipidemias are a risk factor for cardiovascular diseases, especially at a young age. It is known from modern sources that shortchain fatty acids (SCFA) synthesized in the intestine are actively involved in the genesis of dyslipidemia, the spectrum and ratio of which depends on the consumed food substrates. In particular, it has been found that food components such as dietary fiber can affect the lowering of blood lipids by affecting the intestinal microbiota. Therefore, dietary correction can be an important step in the prevention and treatment of dyslipidemia, and as a result, help reduce the risk of developing cardiovascular disease (CD). The aim of the research was to study the content of the main SCFAs (acetate, propionate, butyrate) in the feces of people with dyslipidemia, including taking into account the actual nutrition and consumption of the carbohydrate fraction of the diet (starch, mono- and disaccharides, dietary fiber) as precursors of SCFAs. Material and methods. 70 patients aged 18 to 45 years with dyslipidemia were selected as stool donors. All subjects were divided into 2 groups: the main group with the risk of CD (the risk was determined by the risk scale for CD) and the comparison group with established cardiovascular pathology (CVP). SCFAs in stool samples collected after natural defecation and subjected to immediate freezing at -70 °C were determined by gas chromatography. The diet was analyzed by the 24-hour food recall method. Results. The frequency of occurrence of the optimal ratio acetate - acetic acid : propionate - propionic acid : butyrate - butyric acid (60:20:20) in all groups was no more than 25%. At the same time, in persons at risk of CD, in the SCFA pool there was a pronounced decrease in the proportion of butyrate, which is characterized by cardioprotective properties, up to 15% (with an optimal proportion of 20%) compared with the levels for healthy people noted in the works of M.D. Ardatskaya et al. and A.A. Kurmangulov. And in persons with CVP, the levels of fecal acetate, which is a regulator of metabolic processes, namely lipogenesis, differed insignificantly compared with the values obtained in the studies of the above authors. In all examined individuals, the content of acetate in feces depended on the carbohydrate component of the diet, primarily on the total amount of carbohydrates consumed. And increasing the amount of dietary fiber intake contributed to the elevation of this SCFA. In individuals at risk of CD a significant correlation was found between the content of acetate and the atherogenic index (r=0,695). And in persons with CVP, there was a high negative correlation between the level of acetate in the feces and the amount of mono- and disaccharides in the diet (r=-0,934). Conclusion. In individuals with dyslipidemia and CVD risk factors, the results obtained in this study confirm the need for targeted correction of diets in order to increase the proportion of food substrates, which are potential precursors of butyrate.

[The structure of blood gut microbiota markers in athletes and their relationship with the diet].

It is known that under conditions of ultra-high physical activity and a specific diet, the state of the microbiota plays a significant role in maintaining the health, metabolic and energy status of athletes. The purpose of the study was to evaluate the composition of blood microbial markers in professional football players and physically active people and their correlation with diets in order to substantiate recommendations for their optimization. Material and methods. In a cross-sectional study a group of football players (n=24, 28±3 years old, body mass index - 22.5±1.0 kg/m2) who received a diet according to the training regimen, and a comparison group of physically active individuals (n=25, 34±5 years old, body mass index - 21.8±2.8 kg/m2) have been examined. The method of gas chromatography-mass spectrometry was used to analyze microbial markers of microbiome, mycobiome, virome and blood metabolome populations. Data on actual dietary intake were collected using food diaries for 3 days, followed by data processing with the Nutrium 2.13.0 nutritional computer program. For analysis, individual daily requirements for energy and macronutrients have been calculated based on the basal metabolic rate (according to the Mifflin-San Geor formula, taking into account anthropometric data), the coefficient of physical activity (groups IV and II, respectively). Results. The analysis of the athletes' diet, compared with individual requirements and with the recommendations of the International Society for Sports Nutrition (ISSN), revealed a lack of complex carbohydrates (5±1 instead of 6.1±0.3 g/kg body weight day), an excess of sugars (23±4 instead of <10% of kcal). These figures are significantly higher than the intake of similar nutrients in physically active people in the comparison group. In football players, compared with the comparison group, significant changes in microbial markers were found for Alcaligenes spp., Clostridium ramosum, Coryneform CDC-group XX, Staphylococcus epidermidis (p<0.001), known for their pro-inflammatory activity in the intestine, as well as for Lactobacillus spp. (p<0.001) performing a protective function. In addition, mycobiome markers were increased in athletes: Candida spp. (p<0.001), Aspergillus spp. (p<0.001), among which there are potential pathogens of mycoses. This was not observed in the comparison group. At the same time, an increase in the microbial markers of Alcaligenes spp., Coryneform CDC-group XX, Lactobacillus spp., Streptomyces spp., Candida spp. Micromycetes spp., containing campesterol in the cell wall, in football players positively correlated with a high calorie diet (p<0.001). A similar correlation of mycobiome markers (Micromycetes spp., containing sitosterol in the cell wall, ρ=0.346, p=0.015) was observed with an excess of easily digestible carbohydrates. Taking into account the data obtained, a correction of the diet have been proposed: increasing the consumption of carbohydrates to 7.3-7.5 g/kg of body weight/day by including bakery products from whole grain flour and cereals in the diet (up to 300-370 g/day), limiting simple sugars (up to 90-95 g/day). Conclusion. High physical activity leads to changes in the structure of blood microbial markers, including a shift towards an increase in potentially pathogenic fungi. Wherein, a predictive role is played by an imbalance of macronutrients in terms of quantitative and qualitative composition, an excess of simple sugars, and a lack of slowly digestible carbohydrates. To correct the diet, an additional inclusion in the diet of their main sources - products from cereals (cereals and bakery products) is proposed.

[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.

[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.

[Intestinal microbiote of athletes].

The intestinal microbiota, due to new data on its functions obtained in the last decade, has become a new target point of influence on the organism. However, nowadays knowledge about the possible impact of physical activity and sports on the composition of the gut microbiota and, as a result, on the organism is limited. The aim of this review was to summarize current knowledge about the gut microbiota of healthy people with different levels of physical activity (from athletes to physically inactive people), and to identify patterns in the composition of the microbiota of various surveyed groups. Material and methods. A systematic search was carried out in electronic databases including EMBASE, MEDLINE, Web of Science, Google Scholar and eLIBRARY. The search process was carried out using keywords and logical operators. We included the following studies in our review: a) crossover studies comparing the gut microbiome of subjects with different physical activity; b) studies involving healthy adult women and men (18-45 years old); c) studies written in English and Russian. We excluded studies containing dietary changes, consumption of probiotics or prebiotics, and studies of physical activity in sick people. Results and discussion. Total 743 articles were received, of which 14 articles fully met the search criteria, and 101 articles partially corresponded. An analysis of the data from these studies indicated noticeable differences in the microbiota between athletes and people leading an sedentary lifestyle: the athletes had a greater α-diversity of the microbiota, while the level of microorganisms of the phylum Bacteroidetes was reduced; Akkermansiaceae and Faecalibacterium bacteria are elevated in athletes and people with active lifestyles. Different levels of physical activity in physically active people according to the levels of cardiorespiratory endurance did not affect the level of α- and ß-diversity. When analyzing the effect of loads on the microbiota in various sports disciplines and skill levels, a connection was found with an increase in α-diversity in professionals and highly qualified athletes, with the relative content of series of bacteria (Methanobrevibacter smithii in professional cyclists; Parabacteroides, Phascolarctobacterium, Oscillibacter, Bilophila, Megasphaera in athletes of high martial arts qualifications of wushu; Eubacterium rectale, Polynucleobacter needarius, Faecalibacterium prausnitzii, Bacteroides vulgatus, Gordonibacter massiliensis in athletes of international level of various sports), and certain genera of bacteria have been identified (Parabacteroides, Phascolarctobacterium, Besilibacterium). Conclusion. The data obtained indicate a higher relative proportion of microbiota effective members, which are involved in the fermentation of complex polysaccharides and the production of short-chain fatty acids such as Faecalibacterium prausnitzii, Eubacterium hallii, Phascolarctobacterium, Eubacterium rectale, and Methanobrevibacter smithii, which increases the fermentation efficiency of many bacterial taxa in the gut by using hydrogen gas (H2) and formate to reduce carbon dioxide (CO2) to methane. There is a need to study other members of the microecological community, leading to a better understanding of the adaptation of the gut microbiota to levels of physical activity and its potentially positive effects on metabolism and endurance.

[Minor antibiotics residues in food: what are the risks for consumers].

The problem of contamination of foodstuffs with antibiotic residues does not lose its relevance everywhere, and the most widespread are the quantities of contaminants at the level of regulated values. This raises the concern of specialists in the field of production and processing of livestock products and initiates their appeal to scientific organizations of the hygienic profile for an explanation of the potential health risks associated with the consumption of low doses of antibiotics with food. Material and methods. Analysis and synthesis of data from scientific sources and official documents in the field of assessing health risks when consuming antibiotics with food, with an emphasis on the effects of minor amounts [at the level of sub-inhibitory values below minimum inhibitory concentrations has been carried out. Results and discussion. The issues of direct and indirect human exposure to antibiotics in low doses, including the formation of resistance of intestinal bacteria and the acceleration of the evolution of microbes, accumulation in the organism, the likelihood of allergic reactions, as well as preservation in foodstuffs during heat treatment, have been highlighted. The role of low doses of antibiotics as analogues of biologically active metabolites of bacteria is demonstrated, which, without exerting a toxic effect on the macroorganism, serve as triggers of changes in the microbial ecosystems of humans, animals and habitats through the mechanism of switching on regulation transcription in microbes and activation of horizontal transfer of genes encoding resistance and associated traits. The negativity of the disproportionately wide use of tetracyclines in agriculture, as the cause of the globalization of transferable resistance, has been emphasized, which was justified by the data on the ability of their sub-inhibitory doses to induce the expression of the largest number of mechanisms of its formation and most strongly provoke the horizontal transfer of linked genes between microbes. The need to preserve the current in the EAEU MRL for tetracyclines (0.01 mg/kg product), located in the concentration zone 0.05-0.1 minimum inhibitory concentrations for most sensitive bacteria, safe in terms of resistance induction, has been confirmed. Conclusion. Adequate rationing of antibiotics in food is recognized as a risk management measure for both direct and indirect negative consequences for human health, since the need to ensure MRLs requires manufacturers to strictly adhere to doses, duration of use and withdrawal periods of drugs. This reduce the likelihood of developing resistance in the gastrointestinal tract of animals, the load of the environment by resistant microbes and their transmission along the food chain. The underlying establishment of microbiological acceptable daily intake should be improved by including the selection of intestinal bacteria co-resistance under the influence of a regulated drug, as a marker of the induction of horizontal transfer of resistance genes, in the biological endpoints of determining.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

[Dried fruits marketed in Russia: multi-mycotoxin contamination].

Mycotoxins (MT) - secondary metabolites of micromycetes - are natural contaminants of plant products. Fruits are particularly susceptible to fungal contamination and MT accumulation due to high sugar content. It can occur at any production stage: during vegetation, drying and storage. The most hazardous MT - aflatoxins (AFLs) and ochratoxin A (OTA) - are regulated in dried fruits in some countries. However, their maximum levels (ML) were not set in Russia yet. The present research was aimed at the evaluation of MT contamination of dried fruits marketed in Russia. Material and methods. 54 samples of dried dates (n=11), apricots (n=9), raisins (n=9), prunes (n=7), figs (n=6), apples (n=3) and mixtures for compote (n=9) were analyzed for 32 MTs by HPLC-MS/MS with positive/negative ESI in the MRM mode. Results. OTA and fumonisins (FBs) were the major regulated contaminants, their occurrence proved to be 10 and 17% correspondingly. Emergent metabolites of Fusarium spp. enniatin A (ENN A, 22%) and beauvericin (BEA, 15%); Penicillium spp. mycophenolic and cyclopiazonic acids (MPA and CPA, about 19%); Alternaria spp. tentoxin (TE, 17%) were detected also. Two-thirds of positive samples were contaminated with two and more MTs. All studied samples could be referred as safe within the EU regulations. Conclusion. Some kinds of dry fruits proved to be susceptible to contamination with particular MTs. Characteristic pattern for raisin was the OTA and MPA combination, for figs - FBs and CPA. According to literature data concerning occurrence and safety of MTs and the results of our survey, the long-term monitoring of AFLs and OTA in dry fruits and AFLs, OTA, FBs and CPA in figs from different regions of Russia is necessary to assess the need for setting of MLs of these MTs.

[The role of the gut microbiome in health and diet-related diseases].

In recent years, new data have been obtained on the participation of the organism-associated microbiota in the pathogenesis of many non-communicable diseases. However, these data are mostly multidirectional and require interpretation. The aim - to highlight the role of the intestinal microbiome in the human body in health and some nutritional-dependent pathologies, taking into account modern scientific knowledge. Material and methods. The analysis of domestic and foreign scientific literature in the field of studying the intestinal microbiome in humans and practical measures for its correction in the most common diet-related non-communicable diseases, using the Scopus, Web of Science, PubMed, Google Schoolar, eLibrary, Cyberleninka databases has been held. Results. The data on the significance and function of the intestinal microbiome for the human body in health and in nutrient-dependent diseases, with attention to publications containing information that meets the criteria of scientific evidence have been summarized. It has been shown that the intestinal microbiome plays a biologically significant role not only in digestion processes, but also in many metabolic processes and in the adaptive potential of the human organism. The role of the microbiome in the metabolism of lipids supplied with food, as well as its participation in the mechanisms of development of dyslipidemias and metabolic syndrome in the case of dysbiotic disorders in the intestine, have been described. Conclusion. Nowadays the importance of the intestinal microbiome as an integral factor in the vital functions of the organism that determines the development and maintenance of the immune system, digestive processes, and biochemical balance in humans in norm has been convincingly proven at all levels of numerous studies. However, the study of the effect of dysbiotic changes in the gut microbiota as a direct cause of the development of non-communicable diseases requires further research with a high level of evidence.

[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.

[COVID-19: new challenges for medical science and practical health].

Currently, due to the wide spread of the new coronavirus infection COVID-19 and the need for anti-epidemic measures, medical science should integrate all efforts to ensure, on the one hand, at the global level - the fight against the spread of infection, on the other hand, at the individual level - increasing the adaptive capacity and immune response of the organism to protect against COVID-19. Nutrition is the most important factor determining human health and the functioning of all mechanisms to protect a person from negative environmental factors. For the prevention and treatment of new coronavirus infection COVID-19, a significant role is played by the correction of eating disorders, including vitamin and micronutrient deficiency. Federal Research Centre of Nutrition and Biotechnology, together with Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor), prepared Recommendations on nutrition for children and adults, requiring a self-isolation regime or quarantine at home in connection with COVID-19. In order to promptly inform and advise the population on nutrition optimization in the early days of anti-epidemic measures, an information reference Contact Center was created and is continuously functioning on the basis of the Federal Research Centre of Nutrition and Biotechnology. Equally important is the implementation of measures aimed at the prevention of food contamination with COVID-19 agent. In this regard, the Federal Research Centre of Nutrition and Biotechnology, together with Rospotrebnadzor prepared guidelines on measures to prevent the transmission of a new coronavirus infection through foods. Another aspect that the medical community has faced in connection with the pandemic is the need to reorient specialized medical organizations that have not previously worked with patients with infectious diseases in order to create Clinical centers for the treatment of the new coronavirus infection COVID-19. Thus, in the context of the spread of the new coronavirus infection COVID-19, many areas of medicine are involved in the process of providing anti-epidemic measures. Successful completion of the tasks will significantly reduce the negative consequences of the COVID-19 pandemic for the state and citizens.

[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.

[Methanobrevibacter smithii in irritable bowel syndrome: a clinical and molecular study].

AIM: To assess the role of Methanobrevibacter smithii in patients with irritable bowel syndrome associated with small intestinal bowel overgrowth. MATERIALS AND METHODS: Sixty - seven patients with IBS according to Rome IV were enrolled into the study in whom hydrogen breath test was performed. Thirty - two healthy subjects with negative breath test was used as a control. All IBS symptoms assessed daily with 5 grade Lykert scale for 7 days, stool was assessed by Brystol stool scale. M. smithii was confirmed in stool samples by PCR. RESULTS AND DISCUSSION: In 67 IBS patients CH4 overproduction was found in 32 (47.7%), H2 overproduction in 31 (46.2%) and normal values in 4 (5.9%) by hydrogen breath test. M. smithii was confirmed by stool PCR in all patients with CH4 overproduction. Severity and prevalence of main clinical features of IBS were similar in both SIBO groups but were significantly higher than in control (p.

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.