Сельскохозяйственная биология, 2011, № 4, с. 108-114.

УДК 619+632.9]:579.8

EFFECT OF LONG-TERM ORAL ADMINISTRATION OF THE Bacillus thuringiensis δ-ENDOTOXIN ON MICROFLORA OF LARGE INTESTINE IN MOUSE

E.G. Klimentova¹, A.A. Kuptsova¹, L.K. Kamenek¹, V.V. Guliy²

The authors studied the frequency of distribution of conditional pathogenic microorganisms and hemolytic strains of Escherichia coli and Staphylococcus aureus in microbiocenosis of mouse large intestine during dysbacteriosis, due to the action of Bacillus thuringiensis d-endotoxins. The appearance of conditional pathogenic microorganisms and strains of hemolytic E. coli and S. aureus in large intestine microflora during long-term of use of toxin high doses was revealed.

Keywords: Bacillus thuringiensis δ-endotoxins, intestinal microflora, hemolytic strains of Escherichia coli and Staphylococcus aureus, dysbacteriosis.

Annual analytical studies performed by “Abercade” - the research company focused on industrial market and technologies (Russia, http://www.abercade.ru/about/) – suggest that spore-crystal mixes of Bacillus thuringiensis are the active agents in most of natural insecticides used for pest control in agriculture and forestry.
Parasporal protein crystals formed by different subspecies of B. thuringiensis (so-called cry-proteins or δ-endotoxins) are known to be highly specific pesticides used to combat insects, nematodes, mites (1), along with the fungistatic effect on phytopathogenic fungi (2). Recently, a number of natural insecticides based on purified and activated d-endotoxins of B. thuringiensis has been patented in Russia (Bitiplex, Delta, etc.) that provide a high biological effect on many pests (3). Along with it, cry-genes have been transferred into plants to protect them from harmful insects (Bt-plants).
Cry-proteins can get into the organism of animals and humans per orally (with food), which necessitates evaluation of their effects on microflora in the digestive tract. It has been shown that protein toxins can influence the normal intestinal microbiocenosis. These substances can cause the emergence of new pathogenic and opportunistic strains having potentially unpredictable properties, to change biological characteristics of individual members of the microbiota by selective inhibiting or, alternatively, enhancing their growth, which results in dysbiotic disorders (3). In addition, parasporal proteins of B. thuringiensis have the pronounced antibacterial activity against a number of prokaryotic organisms (4-8).
The purpose of this research was to study the influence of per orally introduced δ-endotoxin of Bacillus thuringiensis on species composition of the microflora in large intestine of warm-blooded animal models.
Technique. The study was performed using B. thuringiensis subsp. kurstaki the strain  Z-52 producing the Cry IA crystals of δ-endotoxin most commonly used in formulations of natural insecticides.  Parasporal crystals were obtained by cultivating the bacteria on agar nutrient media and separating the crystals from spores with p-xylene using a standard technique (9). To remove surface impurities, the crystals were washed with water and 1 M NaCl, and then treated with 0,02 N NaOH for 1 h with stirring to obtain a solution containing a mixture of proteins contained in the crystals. To activate protoxin to the toxin, the solution was incubated with protease of the producent bacteria for 1-2 days at room temperature. After this, protein crystals were precipitated with glacial acetic acid, separated from the supernatant by centrifugation and re-dissolved in 0,02 M phosphate buffer (pH 7,8). Then the solutions with specified protein contents were prepared by diluting with the buffer; the concentration was determined on the spectrophotometer SF-26 (“LOMO”, Russia). The resulting solutions were used for per oral administration to laboratory animals.
 The animal models were 390 white outbreed mice - females with an average live weight of 26 ± 3 g. Housing, keeping, and feeding conditions corresponded to the “Regulationfor Studies using Experimental Animals” (“Pravila provedeniya rabot s ispol’zovaniem jeksperimental’nykh zhivotnykh”, Annex to Decree of the USSR Ministry of Health # 755 of 12.08.1977). Before each series of experiments, the mice were divided into 3 test groups each of 120 individuals. Animals of groups I, II and III were intragastrally administered using a probe with d-endotoxin solution with concentration of, respectively 25, 50 and 100 mg/kg live weight (from 0,0075 to 0,0300 mg per individual). The procedure was performed during 28 days daily at the same time (morning). The control group IV were 30 individuals not given the toxin. The recorded parameters were general health condition and animals’ behavior, feed consumption, fur coat quality, diarrhea or its absence, body weight. To eliminate the effects on the intestinal microflora caused by non-specific factors associated with stress, all control animals received saline instead of δ-endotoxin while being subjected to the same manipulations as the experimental groups. On the 7th, 14th, 21st and 28th day the mice were euthanized with ether, autopsied and the feces were collected from the large intestine contents into a sterile single-use vessel for the investigation of microflora.
Microorganisms were isolated using common bacteriological techniques; analysis and definition were based upon morphological, biochemical and cultural properties (10). Biochemical identification of the cultures was performed using test kits ENTEROtest, STREPTOtest, NEFERMtest, STAPYtest (“Lachema”, Czech Republic). The presence of hemolytic Escherichia was detected by culturing on blood agar (после высева из разведения 1:105 after the inoculation with solution diluted 1:105).
Statistical processing of data was performed using the program StatSoft Statistica 6.0. Reliability of differences between values was considered at p <0,05.
Results. Animals of the control group IV, as well as groups I and II given the toxin solutions at doses of 25-50 mg/kg, showed no symptoms of dysbiosis. The mice were active and consumed all given food completely. In group III, dysbiotic symptoms (low physical activity, bad appetite, swelled stomach and diarrhea) emerged by the end of the 1st week of experiment, and persisted in some individuals during all period of observations. On the 21st day, the number of mice with dysbiosis symptoms has increased by 12 (or 40,0% of the group), by the end of the experiment (on the 28th day) – by 14, or 46,7%.
On the 28th day, from these 14 dysbiotic animals there were revealed by inoculation 255 strains of Gram-positive and Gram-negative opportunistic bacteria and fungi, 58,2% of which were opportunistic enterobacteria (Table 1). From mice of the control group, there were isolated only 192 strains of microorganisms, 52,6% of which were opportunistic Gram-negative bacteria. So, the experimental group III showed higher rate of Gram-negative enterobacteria than the control.

The opportunistic Gram-negative bacteria isolated from group III were represented by predominant E. coli, a significant share of Proteus spp., Klebsiellae spp., Citrobacter spp.; along with it, there were detected representatives of new genera of opportunistic pathogens not found in control - Morganella morganii and Yersinia enterocolitica. Among the opportunistic Gram-positive bacteria isolated from the 14 mice with symptoms of dysbiosis, there was significantly lower number of Staphylococcus epidermidis was than in control, but the larger number of strains of the most pathogenic Staphylococcus aureus. Along with it, there was observed a significant proportion of bacteria the genus Enterococcus. In the control group, there were found mainly Staphylococcus epidermidis and Enterococcus spp. (Table 1).
The established differences in qualitative and quantitative composition of symbiotic microflora in the large intestine allowed to assume the presence of associated taxa of opportunistic pathogens (Staphylococcus-Escherichia), which are more frequently detected in animals that had consumed high doses of d-endotoxin (group III) than in control or in animals of groups I and II given with lower doses.  
Studying the presence of hemolytic strains of Staphylococcus aureus and Escherichia in the large intestine microbiota of mice given a high dose of d-endotoxin (group III), on the 14th and 28th days there was found the greater number of strains of E . coli and S. aureus Hly +, than in the isolates from control mice (Table 2). In groups I-III, the lactose-negative strains of Escherichia were detected more often than in control as well.
Excessive propagation of pathogenic and opportunistic microorganisms (Staphylococcus, Pseudomonas, Klebsiellae, Proteus, Escherichia, Clostridium and fungi) or their associations along with emergence of the large number of hemolytic and lactose-negative Escherichia and Staphylococcus aureus indicate the development of pathological biocenoses in the intestines of  animals fed with high doses of d-endotoxin B. thuringiensis for a long time. This fact is consistent with findings of other authors on the effect of protein toxins in the intestinal microflora (11, 12).

It is believed that toxic proteins firstly affect the normal intestinal microbiota, which is present in the intestine as the biological film preventing penetration of these toxins into the macro-organism. This reduces the population of E. coli with normal enzyme functioning and high antagonistic activity thereby providing the conditions for excessive colonization of lactose-negative and hemolytic strains, whose propagation is normally suppressed by the competitive active symbionts (13).
Thus, the intestinal microflora of warm-blooded animals (white mice) fed for a long time with food containing high doses of δ-endotoxin of Bacillus thuringiensis (100 mg/kg live weight) was found to contain opportunistic enterobacteria, hemolytic and lactose-negative strains of Escherichia coli, which ones were present in larger numbers and detected significantly more often than in intact animals. This fact allows to assume that such doses of δ-endotoxin can be one of the factors contributing to the emergence of these groups of microorganisms in the intestinal microflora (the mechanism of development of the observed dysbiotic disorders requires further study). The obtained data on effects of Cry-proteins on prokaryotic symbionts in the gastro-intestinal tract of warm-blooded animals can be important for evaluating the environmental safety of natural insecticides based on metabolites of B. thuringiensis and transgenic plants synthesizing Cry-proteins.

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