Features of isolation of the anthrax pathogen depending on the type of nutrient medium

Nariman S. Sadykov1, Ramzi N. Nizamov1, Elmira N. Musta ina1, Marina Yu. Gallyamova1, Timur R. Musta in2, Albert K. Galiullin*3, Iva I. Zadorina3, Sergey Yu. Smolentsev4 1Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Scienti ic City-2, 420075, Russia 2LLC, Prom Aseptika, Minskaya st. 35, Kazan city, 420140, Russia 3Kazan State Academy of Veterinary Medicine by N.E. Bauman, Sibirsky tract 35, Kazan city, 420029, Russia 4Mari State University, Lenin Square 1, Yoshkar-Ola city, 424000, Russia

bacteria, anthrax, culture medium, isolation, colony of bacteria ABSTRACT The purpose of the work is to develop a nutrient medium for differentiation of bacillus anthracis from soil aerobic bacilli. In order to achieve the set goal, we used the method of introduction into the environment of cultivation of microorganisms separated from animals and objects of external environment (water, soil, feed, air, scrapes from different surfaces suspected of contamination by their bacillus anthracis) of nutrient substrate -sucrose, used by bacteria of Bacillus genus for synthesis of levan -product of their metabolism, a sign absent in bacillus anthracis. This feature is essential for identi ication and differentiation of bacillus anthracis from closely related saprophytes. To identify and differentiate the bacillus anthracis, the microbes isolated from the external environment were cultivated in a nutrient medium consisting of meat-and-peptone agar (MPA) and stimulator (inductor) synthesis of levan sucrose in the amount of 10% to 100 ml of melted agar. The proposed nutrient media was prepared as follows. Meat-peptonic agar (500 ml) was melted at 100 0 C, 10 g of sucrose was added per 100 ml of medium and after the complete dissolution of sucrose, the nutrient medium was poured into Petri dishes and used for sowing the studied material for identi ication and differentiation of grown crops. The ef iciency of the method has been tested in production experiments with positive evaluation. For this purpose, soil samples taken from the territory of old cattle cemeteries were fractionally sown on MPA and thermostatted for 16-18 hours at 37 0 C and examined crops for the presence of matte and rough (R-form) colonies.

INTRODUCTION
Malignant anthrax is a dangerous infectious disease that has been known for a long time. At the same time, in spite of timely carrying out of antiepidemic and antiepizootic measures, malignant anthrax outbreaks are still observed in almost all countries of the world (Hisao and Masuo, 1989;Brabander et al., 2009). Cases are registered every year not only among animals, but also among humans, including fatal ones. In laboratory diagnosis of malignant anthrax an important role is assigned to indication and identi ication of the causative agent in biological material as well as environmental objects (Viñas et al., 2004).
Of particular scienti ic interest is the development of a nutrient medium for the indication and identiication of bacillus anthracis in veterinary surveillance facilities (Udalova et al., 2015). The analysis of the majority of developed by now nutrient media for indication of differentiation of strains of bacillus anthracis showed, that all of them are peculiar to some or other lacks (Patent RU No. 2289622, IPC S 12 No. 1/20. Pub.20.12.2006, Bul.No. 35;Patent RU No. 2425869, IPC S 12 No. 1/20. Pub.27.05.2003, Bul.No. 10;Patent RU No. 2214453, IPC S 12 No. 1/20. Pub.10.08.2011, Bul.No. 22) (Singh, 2015). In some cases it is the instability of manifestation of differentiating features, such as phosphate formation, lecithinase production and other biochemical features of B.anthracis and closely related aerobic bacilli. In others -it is impossible to perform intraspeci ic differentiation of B.anthracis strains (virulent, avirulent and attenuated) (Carrasco et al., 2008;Mahmoudi et al., 2014;Chilumuru et al., 2015).
Meanwhile, it is known that in the process of their metabolism the bacteria of the genus Bacillus, including B. sereus, B. subtilis, B. mesentericus, B. thuringiencis, B. megaterium, and B. pseudoanthracis, are able to synthesize fruittans -nonrecovering polysaccharides (levan). Synthesis of levans is carried out by an enzyme -levansaccharose. When growing in a sugar-rich environment, the bacterium releases enzyme, which splits sucrose into glucose and fructose. All glucose is consumed by the microorganism, while fructose is polymerized by the enzyme to form levane. Therefore, the synthesis of levans on the cultivation medium of the tested microorganisms can serve as one of the most important taxonomic (differential) features of closely related soil aerobic bacilli.
Lack of effective nutrient media for indication and differentiation of bacillus anthracis determines the relevance of this work.
Taking into account the proposed, the aim of the work is to develop a nutrient medium for differentiation of bacillus anthracis from soil aerobic bacilli.
The nutrient medium was prepared from dry components. The weighted amount of the dry medium were dissolved in distilled water, brought to a boil when stirring, boiled for 2-3 minutes, cooled to (40±5) 0 C and poured into sterile Petri dishes or bacteriological tubes.
Microbiological studies were conducted with 3 strains of bacillus anthracis differing in virulent properties, 6 strains of soil aerobic bacilli and 3 strains of attenuated vaccines. The obtained experimental data were statistically processed using Microsoft Of ice Excel 2003, "Statistica 6.0" PC software.

RESULTS AND DISCUSSION
Today, as a universal nutrient medium for both the indication and differentiation of bacillus anthracis and the etiological agent of other dangerous infections (SDI), MPA is used, the source of nitrogen in which are organic substances of meat water, products of protein separation (peptones); MPA has been known for more than 80 years and still remains the basic medium used in clinical microbiology in different countries for the purposes of sowing, cultivation and subsequent identi ication, provided various additives are used. In English-speaking countries the environment is known as Beef EXTRACT AGAR.
One of the promising trends in the development of rapid detection and differentiation of infectious agents is considered to be enzymatic, associated with the presence of the microorganism of certain biochemical properties that distinguish it from other representatives of this genus. This direction can be realized through the use of differentialdiagnostic nutrient media, which include: a nutrient base that provides bacterial growth; a chemical substrate, the relation to which is a diagnostic attribute for a given microorganism.
In comparative assessment of the enzymatic activity of B. anthracis strains and closely related aerobic bacilli, we found a difference in their ability to synthesize fruittansβ-2.6-bound polysaccharides -levans with the enzyme -levansaccharose. This feature made it possible to carry out research to develop a dense diagnostic nutrient medium for simultaneous isolation and differentiation of anthrax cultures and spore-forming saprophytes close to bacillus anthracis.
In the course of the works the composition of the nutrient medium providing optimal conditions for growth and manifestation of biochemical activity of the microorganisms under test was selected experimentally, substrate concentrations were determined.
The proposed nutrient medium was prepared as follows. Meat-peptone agar, contained in a glass tube of 500 ml, melted in a water bath at 100 0 C, 10 grams of sucrose were added per 100 ml of medium. After complete dissolution of sugar (sucrose), the resulting nutrient medium (100 ml MPA + 10% sucrose) was poured into Petri dishes, placed in a thermostat at 37 0 C for 30 minutes for drying and release from condensation. The nutrient medium spilled into Petri dishes after drying is used for sowing the tested material for identi ication and differentiation of grown crops.
The nutrient medium prepared according to the method described above is sown fractionally with three dishes per culture: anthracic virulent culture B.anthracis culture, anthracic vaccine strains B.anthracis 55, attenuated capsule strain B.anthracis M-71, closely related saprophytes: B.sereus, B.subtilis, B.mesentericus, B.megaterium, B.pseudoanthracis. The crops were thermostatted at 37 0 C for 16-18 hours. At the end of this time visual analysis of the grown crops was carried out.
The results of viewing the grown cultures showed that the dishes with the test medium in which malignant anthrax strains were sown showed abundant growth of typical anthrax cultures with a matte tint in all three dishes, although fractional sowing was performed.
In crops of closely related saprophytes: B.sereus, B.subtilis, B.mesentericus, B.megaterium, B.pseudoanthracis revealed a similar abundant growth of cultures throughout the whole dish and on the surface of cultures was found an abundant mucous substance in the form of droplets -levane, a product of synthesis of saprophytes close to bacillus anthracis.
The multiple repetitions of similar tests yielded identical results. Therefore, the appearance on the surface of the studied (suspicious for malignant anthrax) cultures of abundant mucous substancelevane, testi ies to their belonging to the Bacillus genus, but not to the species of B.anthracis (bacillus anthracis).
Thus it is established that the kind of B.anthracis irrespective of a degree of virulence, ability to capsule formation or its absence, on the used nutrient medium does not form mucous substancelevan that is a differential criterion of an accessory of investigated cultures to B.anthracis (bacillus anthracis).
Nutrient media with the above component ratios were sown with virulent and vaccine (B.anthracis 55, STI-1) strains of bacillus anthracis, the crops were examined as described above.
It was established that with medium with minimal sucrose concentration (5%) the biomass yield was 2.1 billion in 1 ml, and with medium with both maximum (15, 20%) and optimal (10%) sucrose concentration the biomass yield was 2.5 billion / B.anthracis ml.
Consequently, an increase in sucrose concentration does not lead to an increase in biomass, so the optimal ratio of components in the proposed nutrient medium MPA -90% and sucrose -10%.
In the next series of experiments we tested the ef iciency of the nutrient medium at indication and identi ication of bacillus anthracis in external environment objects.
The bacillus anthracis gets into the soil, goes through a long period of time in spore form and poses a serious problem for veterinary medicine, as the presence of the agent at the burial site of malignant anthrax corpses poses a threat of outbreak of this particularly dangerous infection. Therefore, the detection of the agent in the soil and its neutralization is the primary task of preventing this dangerous sapronosis. However, the release of the agent in the soil is associated with great dif iculties due, irstly, to the insigni icant concentration of the agent in the soil and, secondly, to the presence of aerobic bacilli closely related to bacillus anthracis in large quantities: B.sereus, B.subtilis, B.mesentericus, B.megaterium, B.pseudoanthracis, which by their cultural and morphological properties are similar to bacillus anthracis and which, by persisting in soil with the speci ied saprophytes, acquires similar properties (avirulence, acapsulogenicity, mobility, etc.). Under these conditions, existing methods and means of identi ication and differentiation of the agent lose effectiveness.
With this in mind, we conducted experiments on the isolation and identi ication of bacillus anthracis using the proposed nutrient medium.
For this purpose, soil samples taken from the territory of the cattle dumps, after treatment in accordance with the "Instruction on the separation of bacillus anthracis in the objects of veterinary inspection" were fractionally sown on MPA, thermostatted 16-18 hours at 37 0 C and looked through the crops for the presence of matte and rough (R-shaped) colonies. Suspicious of malignant anthrax crops in the colony were transferred to the proposed nutrient medium, followed by thermostatting and crop viewing as described above.
It was found that out of 106 colonies selected, suspected of malignant anthrax, only 2 colonies gave abundant growth of microbes on the tested medium without the formation of abundant mucous matter -levane, while cultures from 104 colonies grown from samples of cattle-breeding soil, on the tested medium formed abundant mucous matter -levane, which indicates their belonging to the Bacillus genus and species B.B.subtilis, B.mesentericus, B.megaterium, B.pseudoanthracis which are always present in soil as well as in plants.

CONCLUSIONS
Having carried out purposeful researches on construction of differential-diagnostic nutrient medium for microbiological researches, and in particular, for indication and differentiation of bacillus anthracis from close to bacillus anthracis soil aerobic bacilli, the composition of nutrient medium providing optimal conditions for growth and manifestation of biochemical activity of microorganisms under test was experimentally selected. It has been found that the best results can be obtained by using meat-peptone agar (MPA) as the basis for the nutrient medium. The optimal concentration in the sucrose substrate medium was 10%.
In this way, the feed medium offered allows, 1. Signi icantly accelerate the time of bacillus anthracis excretion and identi ication from environmental objects; 2. Reliably differentiate bacillus anthracis from closely related aerobic saprophyte bacilli: B.sereus, B.subtilis, B.mesentericus, B.megaterium, B.pseudoanthracis species without using special nutrient media (GCI, hydrogen carbonate water, MPA with blood), expensive diagnostics (enzyme and luorochromic antibodies), infection of susceptible animals, because these tests fully con irmed the results of identi ication of bacillus anthracis using the proposed sugarcane nutrient media.
The distinctive features of the proposed nutrient medium are, 1. Using meat-pepton agar as the basis for the nutrient medium of solid nutrient substrate; 2. Using sucrose as a stimulant for the synthesis of levan; 3. Exclusion of expensive Henks solution, blood serum and blood agar used in known nutrient media; 4. There is no need to study the capsule formation of the obtained crop using the described reagents and equipment, because due to the ecological interaction of bacillus anthracis with extreme factors (pH, chemical substrates, aerobic saprophytes, etc.) of soil and other objects, the capsulogenicity of the etiological agent may be temporarily or permanently absent.