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.

A Method of Direct Quantitation of Lactobacillus spp. in Intestinal Contents Based on Real-Time PCR.

The method of direct quantitation of Lactobacillus spp. and L. acidophilus in intestinal contents based on real-time PCR was developed. It does not require culturing and allows estimating the number of living lactobacilli cells (measured in lg CFU) in absolute quantitative PCR format.

[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.

[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.

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

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

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

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