TOXIN PRODUCTION ABILITY OF BACILLIUS CEREUS STRAINS FROM FOOD PRODUCT OF UKRAINE

Potential pathogens of foodborne toxic infections – bacterial contaminants Bacillus cereus isolated from plant raw materials and food products from the Ukrainian region were investigated. When determining of the proportion of isolated bacilli from the plant samples, it was established that the epidemiologically significant microorganisms of Bacillus cereus as agents of food poisoning are the second largest. The average value of contaminated samples of Ukrainian plant raw materials and processed products with Bacillus cereus is 36.2 %. The ability of Bacillus cereus strains identified by a complex of morphological, tinctorial, cultural and biochemical properties, to produce specific emetic and enterotoxins was studied. Molecular genetic diagnosis and detection of the toxin-producing ability of isolated 42 Bacillus cereus strains showed both the possibility of their rapid identification and the presence of specific toxicity genes. Multiplex polymerase chain reaction (PCR) was carried out with specific primers to detect toxicity determined of various bacilli genes: nheA, hblD, cytK, cesВ. The distribution of toxigenic genes is significantly different among the Bacillus cereus isolates from various sources. The nheA, hblD and cytK enterotoxin genes were detected in 100, 83.3 and 61.9 % of the investigated strains of Bacillus cereus, respectively. The cesB gene encoding emetic toxin was detected in 4.8 % of strains. Molecular-genetic PCR-method confirmed that all the isolated strains belong to the Bacillus cereus group, and the ability to produce toxins can be attributed to five groups. The main toxins that produce the investigated Bacillus cereus strains were nhe and hbl enterotoxins encoded by the corresponding genes of nheA and hblD. The enterotoxic type of Bacillus cereus was predominant in Ukrainian region. Studies of domestic plant food raw materials and products have confirmed the need to improve microbiological control of product safety by introducing accelerated specific diagnostics of contaminants by molecular genetics methods.


Introduction. Formulation of the problem
Regulated methods of diagnosing the safety of food and raw materials are classical methods of food microbiology, which are time-taking, based on the phenotypic characteristics of microorganisms and are not always able to diagnose their toxigenic properties.
Volume 11 Issue 3 / 2017 Analytical information on the inaccuracy of indication of bacillary food poisoning, the need for a preventive analysis of the risks that aerobic and facultativeanaerobic spore-forming microorganisms of the genus Bacillus, cause the urgency of their detection by accelerated modern methods.Such diagnostics will allow to produce new competitive food of guaranteed quality and microbiological safety [1,2].

Analysis of Literature
Food poisoning caused by the presence of Bacillus cereus in foodstuffs is recorded in almost all countries [3,4].According to the Center for Disease Control and Prevention (CDC Foodborne Outbreak Online Database), more than 60000 cases of diseases caused by B. cereus are recorded annually in the United States.Bacillus cereus, a rod shapes, gram-positive, spore-forming food pathogen, play an important role as the causative agent of diarrheal and emetic types of food poisoning [3].The diarrheal type of food poisoning is caused by heat-labile enterotoxins such as hemolysin BL (hbl), nonhemolytic entrotoxin (nhe) and cytotoxin K (cytK).The hbl-and nhe-complex both consist of three proteins (tripatite toxins).Cytotoxin K is a pore forming toxin cause necrotic enterotitis.The diarrheal syndrome, including abdominal pain and diarrheal symptoms, appears 8 to 16 h after ingestion of contaminated food.The emetic syndrome, which is characterized by nausea and vomiting within 1 to 5 h after ingestion of contaminated food, is causes by emetic toxin cereulide, a depsipeptide structurally related to potassium ionophore valinomycin, which is produced by a nonribosomal peptide synthetase (NRPS) and coded cesB gene [4].
Bacillus cereus can cause people a wide range of diseases, including food poisoning, systemic and local purulent infections, including Lightning sepsis, meningitis, brain abscess, endophthalmitis, pneumonia, endocarditis, osteomyelitis, skin gas gangrene infection, etc., and mastitis of cattle in animals.It is noted that some patients with vomiting symptoms with bacillary food infection are erroneously diagnosed with an intoxication syndrome caused by Staphylococcus aureus, whereas the false diarrhea-causative agent of this toxicoinfection is Clostridium perfringens [2][3][4][5].
Concerning the methods for the determination of Bacillus cereus, it is known that the characteristics of metabolic properties of the pathogen are often used as identification tests, which are part of standardized methods of analysis, and this does not always allow a clear differentiation of pathogenic agents from nonpathogenic, phenotypic-like pathogens [5].Bacillus cereus group was divided into emetic-and enterotoxinproducing strains, but emetic toxin-producing B. cereus is difficult to detect immunochemically [6].This reduces the probability of the results of the analysis, complicates the assessment of the prevalence of pathogens in food and raw materials and does not guarantee the unjustified defects of products.
The aim of this study was to identify and detect entero-and emetic toxin-producing bacteria among Bacillus cereus strains, isolated from Ukrainian food plant raw materials and products.
To achieve this aim, you must accomplish the following tasks: 1) to determine the species composition of bacilli contaminant isolated from Ukrainian food plant raw materials and products; 2) to establish the contamination of samples of plant raw materials and products of its processing with epidemiologically significant microorganisms of B. cereus; 3) to detect the genes of toxicity among investigated B. cereus strains; 4) to identify the major toxins among B. cereus from Ukrainian region.

Research Materials and Methods
The widespread and industrially grown kinds of vegetables, fruits, berries, in particular, green peas, beetroot, tomatoes, carrots, apples, pears, plums, peaches, dill, spinach, parsley, strawberry, a number of canned and dried products, and also spices have been investigated [5,7].Samples of tested materials were selected according to standardized selection rules for the average sample [8,9].
The reference strain B. cereus ATCC 11778 and 42 bacilli strains isolated from food plant raw materials and products, and according to the results of previous studies, identified as B. cereus by studying their morphological, tinctorial, physiological and biochemical characteristics and fatty acid composition of cells [10].Also in the study used collections bacilli strains: B. cereus UKM В-5671, Paenibacillus polymyxa B-5760 T , P. macerans B-5803 T .
Samples of food for PCR were prepared by the priority method developed by us [10].Multiplex PCR was performed using specific primers to bacilli sequences according to Zhang et al. [11].DNA was isolated from the samples using the SureFast® PREP Bacteria F1021 (CONGEN, Germany).The following 4 pairs of specific oligonucleotide primers for the toxicity genes were used.Also the following pairs of specific oligonucleotide primers for the groEL gene were used which is characteristic of all strains of the Bacillus cereus group (Table 1).
PCR cycles are are primary denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min, elongation at 72 °C for 1 min, final elongation at 72 °C for 7 min (Thermal cycler with BioRad software, USA).Primers were chosen on the basis of literature data [11][12][13] and synthesized by SPC "Simesta VAAL" (Odessa, Ukraine).Composition of the mixture for PCR: supermix -10 μl, specific olygonucleotide primers for the toxicity genes -6 μl, DNA -2 μl, H 2 O -2 μl, amount of PCR mixture -20 μl.As a negative control PCR-mixture without DNA was used.Electrophoresis of PCR products Volume 11 Issue 3 / 2017 was carried out in a 1.5% agarose gel.Trisacetate buffer was used (Equipment for electrophoresis of PCR products from BioRad, USA).DNA was stained with ethidium bromide (0.5 μg/ml) and photographed with a video system (BioRad, USA) under UV light (wavelength 312 nm).A visual evaluation of the size of the formed amplicons was carried out using molecular weight markers (pBR322/BsuRI, Fermentas, Latvia).
The bacillary contaminants of the investigated samples are given in Table 2; the Subtilis-licheniformis group in Ukrainian food plant raw materials and products is the most numerous one.By determining of the proportion of isolated bacilli from the plant samples, it was established that the epidemiologically significant microorganisms of B. cereus as agents of food poisoning are the second largest.
The obtained results allow us to estimate the essential component of the epiphytic microbiota of plant material, which forms the so-called residual microbiota of products of its processing.According to a number of researches [2-5, 10,12], the control of semi-finished products and finished products is based on the determination of the presence and number of these microorganisms.The microbiota of plant material is diverse, but the micellar and non-mecidal mushrooms in thermally processed products are less dangerous to the consumer than spore-forming bacteria [5].The prevailing number of bacilli -potential pathogens of food spoilage, among which the possible presence of pathogenic species (B.cereus), makes it urgent to search for accelerated and expressive methods for their diagnosis.In literary sources, we did not find systematic information about the microbiota of plants isolated in Ukraine; therefore the given results are new and necessary from the point of view of their practical use.

The results of the research and their discussion
During the production of canned products, the main source of infection of B. cereus serves as the main raw material and auxiliary materials [4,5].Since microorganisms in this group cause foodborne diseases and are potentially enterotoxic to humans, the ability to quickly detect B. cereus in plant material is crucial.Data in table 3 shows the quantitative characteristics of contaminated B. cereus plant material, which is processed industrially.Comparing the results with those given for plants from the city of Mexico, it is possible to note practically the same trends of detection of B. cereus -50.0 % and 57.0 % for the Ukrainian and Mexican regions, respectively [4].The average value of contami-Volume 11 Issue 3 / 2017 nated samples of Ukrainian plant raw materials and processed products with B. cereus is 36.2%.
Percentage of strains containing enterotoxin genes nheA, hblD and cytK among investigated B. ce-reus strains was 100, 83.3 and 61.9 %, respectively.The cesB gene encoding emetic toxin was detected in 4.8 % of strains (Table 4).The results suggest that the examined dried products, fruit and berries were free of the emetic toxin but not free of enterotoxins and the distribution of enterotoxic genes is significantly different among the B. cereus isolates from various sources.All investigated strains of B. cereus were divided into 5 groups according to the presence or absence of toxic genes (Table 5).Only 2 strains from group I (4.8 %) have to ability to cause both diarrheal and emetic type of food poisoning.Group II (7 strains, 16.6 %) contained the nheA, hblD and cytK enterotoxin genes, but no cesB encoded emetic toxin.Group V was the major patterns and represented 38.1 % strains.The reference strain B. cereus ATCC 11778 has all the tested genes of toxicity.
Figure 1 shows the electrophoregram of PCR products of some strains of bacilli with specific oligonucleotide primers to the groEL gene, which is characteristic for most representatives of the Bacillus cereus group, and 4 toxic genes: nheA, hblD, cytK, cesB.B. cereus П90-1 (from carrot) and B. cereus П90-4 (from zucchini) contain only the nheA toxic gene and belong to the greatest group V. B. cereus П90-9 (from eggplants) from group III has 2 genes of toxicity: nheA and hblD.Reference strain B. cereus UKM В-5671 forms only the amplicon size 400 bp to the groEL gene.For the use of DNA of Paenibacillus polymyxa and P. macerans no amplification product was obtained.
These finding revealed that nhe and hbl enterotoxins encoded by nheA and hblD genes were the major toxins among B. cereus investigated in this study and enterotoxic type of B. cereus was predominant in Ukrainian region.