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.

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.

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.

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.

[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.

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.

[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.

[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.

[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.